ES2978295T3 - Modified recombinant viral vectors with tropism and their uses for the targeted introduction of genetic material into human cells - Google Patents

Abstract

Provided herein are compositions and methods for retargeting recombinant viral capsid proteins/capsids/vectors, e.g., in vivo, with a multispecific binding molecule, such as a bispecific antibody, that specifically binds to a heterologous epitope displayed by the capsid protein and a protein expressed in the cell of interest for targeted delivery of a nucleotide of interest. (Automatic translation with Google Translate, no legal value)

Description

DESCRIPTION

Modified recombinant viral vectors with tropism and their uses for the targeted introduction of genetic material into human cells

The sequence listing written in file 10335WO01_ST25.txt which is 88 kilobytes in size, was created on June 27, 2018.

Technical field

The disclosure herein relates generally to tropism-modified recombinant adenovirus-associated virus (AAV) vectors and compositions comprising the same, which are useful for the targeted introduction of genetic material into cells and/or tissues.

Background of the invention

Gene delivery to particular target cells has emerged as one of the most important technologies in modern medicine for diagnosis and gene therapy of a variety of genetic and chronic diseases. So far, progress in the clinical application of gene therapy has been limited by the lack of ideal gene delivery vehicles. To achieve therapeutic success, gene delivery vehicles must be capable of transducing target cells while avoiding transduction of non-target cells. Specifically, when the natural tropism of the virus does not meet the instant therapeutic needs, there is a need for recombinant viral vectors where the natural tropism is abrogated or diminished and the desired tropism is successfully engineered. (Buchholzet al.).

In recent years, most progress in vector development has been made using naked viruses (e.g., viruses comprising a capsid formed by viral capsid proteins without an envelope (e.g., lipid bilayer)) such as adeno-associated viruses (AAV) and adenoviruses (Ads), as well as enveloped viruses (e.g., viruses whose capsid is surrounded by a lipid bilayer) such as retroviruses, lentiviruses, and herpes simplex viruses. AAV-based vectors have been the focus of much research, since AAVs are non-enveloped viruses that are only weakly immunogenic but capable of transducing a wide range of species and tissues in vivo without evidence of toxicity.

AAVs are small, nonenveloped, single-stranded DNA viruses. The AAV genome is 4.7 kb and is characterized by two inverted terminal repeats (ITRs) and two open reading frames encoding the Rep and Cap proteins, respectively. The two ITRs are the only cis elements essential for AAV replication, packaging, and integration. The Rep reading frame encodes four proteins of molecular weight 78 kD, 68 kD, 52 kD, and 40 kD. These proteins function primarily in regulating AAV replication and AAV integration into host cell chromosomes. The Cap reading frame encodes three structural (capsid) viral proteins (VPs) that have molecular weights of 83–85 kD (VP1), 72–73 kD (VP2), and 61–62 kD (VP3). More than 80% of the total AAV virion proteins comprise VP3; In mature virions VP1, VP2 and VP3 are present in a relative abundance of approximately 1:1:10. In vitro, all three proteins spontaneously assemble into virion-like structures, i.e. viral capsids. It therefore appears that viral capsid formation in infected cells occurs independently of viral DNA synthesis (reviewed by Kotinet al. (1994) Hum. Gene Ther. 5:793).

Among all known AAV serotypes, AAV2 is perhaps the best characterized serotype, because its infectious clone was the first to be produced (Samulski et al. (1982) Proc. Natl. Acad. Sci. USA 79:2077-2081). Subsequently, the complete sequences for AAV3A, AAV3B, AAV4, and AAV6 have also been determined (Rutledge et al. (1998) J. Virol. 72:309-319; Chiorinie et al. (1997) J. Virol. 71:6823-6833; S. Muramatsu et al. (1996) Virol. 221:208-217). Generally, all AAVs share greater than 80% nucleotide sequence identity.

AAV is a promising vector for human gene therapy since, unlike other viral vectors, AAVs have not been shown to be associated with any known human diseases and are not generally considered to be pathogenic (Muzyczka et al. (1992) Current Topics in Microbiology and Immunology 158:97-129). Furthermore, AAV can safely transduce postmitotic tissues with relatively low immunogenicity and is able to integrate into host chromosomes in a site-specific manner and into tissue cultured cells on chromosome 19 if Rep proteins are delivered in trans. (Kotinet al.(1990)Proc. Natl. Acad. Sci.USA 87:2211-2215; Samulskiet al.(1991)EMBO J.10(12):3941-3950; Balagueet al.(1997)J. Virol.71:3299-3306; Suroskyet al.(1997)J Virol.71:795 1-7959). Integrated AAV genomes have been shown to allow long-lasting gene expression in several tissues, including muscle, liver, and brain (Fisher (1997)Nature Med.3(3):306-312; Snyder et al.(1997)Nature Genetics16:270-276; Xiao et al.(1997)Experimental Neurology144:113-124; Xiao et al.(1996)J. Virol.70(11):8098-8108).

Several viruses, including AAVs, infect cells through a virus/ligand:cell/receptor interaction, ultimately resulting in endocytosis of the virus by the infected cell. This ligand:receptor interaction is the focus of much research on viral vectors, for example, it can be manipulated to redirect the natural tropism of a virus from a naturally permissive cell to infection by the wild-type virus to a target cell, for example, through a receptor expressed by the target cell.

In theory, redirecting a vector toward any cell surface protein or marker should result in infection of a target cell, as most cell surface receptors or markers are involved in endocytosis pathways, either constitutive (e.g., for recycling) or ligand-induced (e.g., receptor-mediated). These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome where the receptors are sorted, and are then recycled to the cell surface, stored intracellularly, or degraded in lysosomes. As such, platforms for redirecting viral vectors often aim to abrogate the natural tropism of the viral vector and redirect the viral vector to a receptor or marker expressed solely or primarily by the target cell. Many of the advances in targeted gene therapy using viral vectors can be summarized as non-recombinatorial (non-genetic) or recombinant (genetic) modifications of the viral vector, resulting in pseudotyping, expansion and/or retargeting of the natural tropism of the viral vector. (Reviewed in Nicklin and Baker (2002) Curr. Gene Ther. 2:273-93; Verheiji and Rottier (2012) Virol Advances 2012:1-15).

Non-genetic approaches typically utilize an adaptor, which recognizes both a wild-type (unmodified) virus surface protein and a target cell. Soluble pseudoreceptors (for wild-type virus), polymers such as polyethylene glycol, and antibodies or portions thereof have been used as the virus-binding domain of adaptors, while natural peptide or vitamin ligands, and antibodies and portions thereof have been used for the cell-binding domain of the adaptors described above. With this approach, retargeting of the viral vector to a target cell can be achieved by binding of the vector:adapter complex to a protein expressed on the surface of the target cell, e.g., a cell surface protein.

This approach has been used for AAV (Bartlettet al.(1999)Nat. Biotechnol.74: 2777-2785), adenovirus (Hemminkiet al.(2001)Cancer Res.61: 6377-81; van Beusechemet al.(2003)Gene Therapy10:1982-1991; Einfeld,et al(2001,J. Virol.75:11284-91; Glasgowet al.(2009)PLOS One4:e8355), herpesviruses (Nakanoet al.(2005)Mol. Ther.11:617 -24), and paramyxovirus (Bianet al.(2005)Cancer Gene Ther.12:295-303; Bianet al.(2005)Int. J. Oncol.29:1359-69), Coronavirus (Haijemaet al.(2003)J. Virol.77:4528-43]8; Wurdingeret al.(2005)Gene Therapy12:1394-1404).

A more popular approach has been the recombinant genetic modification of viral capsid proteins and hence the viral capsid surface. In indirect recombinant approaches, a viral capsid is modified with a heterologous "scaffold", which is then linked to an adaptor. The adaptor binds to the scaffold and the target cell. (Arnold et al. (2006) Mol. Ther. 5:125-132; Ponnazhagenet al. (2002) J. Virol. 76:12900-907; see also WO 97/05266). Scaffolds such as (1) Fc-binding molecules (e.g., Fc receptors, Protein A, etc.), which bind to the Fc of antibody adaptors, (2) (strept)avidin, which binds to biotinylated adaptors, (3) biotin, which binds to adaptors fused to (strept)avidin, and (4) protein:protein binding pairs that form isometric peptide bonds such as SpyCatcher, which binds a SpyTagged adaptor, have been incorporated into Ad (Pereboeva et al. (2007) Gene Therapy 14: 627-637; Park et al. (2008) Biochemical and Biophysical Research Communications 366: 769-774; Henning et al. (2002) Human Gene Therapy 13:1427-1439; Banerjee et al. (2011) Bioorganic and Medicinal Chemistry Letters21:4985-4988), AAV (Gigoutet al.(2005)Molecular Therapy11:856-865; Stachleret al.(2008)Molecular Therapy16:1467-1473), and togavirus (Quetglaset al.(2010)Virus Research153:179-196; Ohnoet al.(1997)Nature Bio technology15:763-767; Klimstraet al.(2005)Virology338:9-21).

In a direct recombinant targeting approach, a targeting ligand is directly inserted into, or coupled to, a viral capsid, i.e., proteinaceous viral capsids are modified to express a heterologous targeting ligand. The ligand is then retargeted, e.g., it binds to, a receptor or marker expressed preferentially or exclusively on a target cell. (Stachler et al.(2006)Gene Ther.13:926-931; White et al.(2004)Circulation109:513-519.). Direct recombinant approaches have been used in AAV (Parket al.,(2007)Frontiers in Bioscience13:2653-59; Girodet al.(1999)Nature Medicine5:1052-56; Grifmanet al.(2001)Molecular Therapy3:964-75; Shiet al.(2001)Human Gene Therapy12:1697-1711; Shi and Bartlett (2003)Molecular Therapy7:515-525), retroviruses (Dalbaet al. Current Gene Therapy5:655-667; Tai and Kasahara (2008)Frontiers in Bioscience13:3083-3095; Russell and Cosset (1999)Journal of Gene Medicine1:300-311; Erlweinet al.(2002)Virology302:333-341; Chadwicket al.(1999)Journal of Molecular Biology285:485-494; Pizzatoet al.(2001)Gene Therapy8:1088-1096), poxviruses (Guseet al.(2011)Expert Opinion on Biological Therapy11:595-608; Galmicheet al.(1997)Journal of General Virology78:3019-3027; Paulet al.(2007)Viral Immunology20:664-671), paramyxoviruses (Naka mura and Russell (2004)Expert Opinion on Biological Therapy4:1685-1692; Hammondet al.(2001)Journal of Virology75:2087-2096; (2010)Clinical Pharmacology and Therapeutics88:620-625; Blechacz and Russell (2008)Current Gene Therapy8:162-175; Russell and Peng (2009)Current Topics in Microbiology and Immunology330:213-241), and herpesviruses (Shah and Breakefield (2006)Current Gene Therapy6:361-370; Campadelli-Fiumeet al.(2011)Reviews in Medical Virology21:213-226).

Each of the three approaches has advantages and disadvantages. A major advantage of a direct recombinant approach is that viral specificity is inherent to the viral genome and can be maintained upon replication. However, for this and indirect recombinant approaches, the ability to genetically modify the virus requires that the capsid structure be maintained, and that the targeting ligand or scaffold be placed in a position that properly tolerates and displays the targeting ligand or scaffold, thus limiting the repertoire of appropriate ligands or scaffolds that can be used. As such, recombinant targeting methods are limited by naturally occurring molecules useful as targeting ligands, leading to the incorporation of other binding ligands such as antibodies or portions thereof. Both nonrecombinatorial and recombinant adapter platforms are advantageous because of the flexibility of the adapter used. However, it is difficult to achieve optimal transduction efficiencies with these two-component systems.

Also mentioned is Wickhamet al. (1997)Journal of Virology,71(10): 7663-7669 which deals with adenovirus-mediated gene delivery targeting T lymphocytes via CD3.

Provided herein is an AAV retargeting strategy that overcomes problems inherent to previous recombinant retargeting strategies by inserting a heterologous epitope into an AAV capsid. The instant recombinant AAV capsid exhibits reduced to abolished natural tropism, which is restored and retargeted upon combination with a multispecific, optionally bispecific, binding molecule comprising an antibody paratope, e.g., Fv, that specifically binds to the heterologous epitope and a retargeting ligand that specifically binds to a target cell, particularly within certain ratios of viral vector:multispecific binding molecules.

Summary of the invention

Disclosed herein are recombinant adeno-associated virus (AAV) capsid proteins, AAV capsids comprising the recombinant AAV capsid proteins, AAV vectors comprising the nucleotide of interest encapsulated by the recombinant AAV capsid; wherein said AAV capsid proteins, AAV capsids and AAV vectors are genetically modified to comprise (display) a heterologous epitope, wherein the heterologous epitope (part thereof or together with the AAV capsid protein) forms a binding pair with an antibody paratope, and wherein the recombinant AAV capsid protein/capsid/vector may further comprise a mutation, insertion or deletion at an amino acid position involved in receptor binding, e.g., the (natural) tropism of the AAV capsid protein/capsid/vector, such that the recombinant AAV capsid protein or an AAV capsid comprising the recombinant AAV protein has a reduced to abolished (natural) tropism (e.g., has, in the absence of a multispecific, optionally bispecific binding molecule, a transduction efficiency that is lower than the transduction efficiency of a reference AAV capsid protein/capsid/vector lacking the heterologous epitope, or undetectable transduction efficiency in the absence of a multispecific, optionally bispecific, binding molecule). Such reduced to abolished (natural) tropism of the recombinant AAV capsid protein/capsid/vector may be enhanced or restored in the presence of an appropriate multispecific, optionally bispecific, binding moiety. Accordingly, also described are compositions comprising (1) recombinant AAV vectors having a capsid comprising a recombinant AAV capsid protein described herein and (2) a multispecific, optionally bispecific binding molecule comprising the antibody paratope and a targeting ligand, including compositions comprising certain ratios of viral vector:multispecific binding molecule; and also described herein are uses thereof for directing and/or introducing genetic material into a target cell. Also described herein are methods for retargeting a recombinant AAV vector, e.g., for targeted delivery of a nucleotide of interest to a target cell, comprising contacting the recombinant AAV vector with a multispecific, optionally bispecific, binding molecule, and methods for producing the recombinant AAV vector are also described.

Accordingly, the present invention provides a composition comprising:

(i) a recombinant adeno-associated virus (AAV) vector comprising an AAV capsid encapsulating a nucleotide of interest,

wherein the AAV capsid comprises a recombinant AAV capsid protein modified to comprise a heterologous epitope, wherein the recombinant AAV capsid protein is derived from an AAV capsid gene modified to express the heterologous epitope;

(ii) a multispecific binding molecule comprising

(a) an antibody paratope that specifically binds to the heterologous epitope; and

(b) a retargeting ligand that specifically binds to a protein or cell surface marker; and optionally

(iii) a pharmaceutically acceptable carrier,

where the AAV capsid presents:

- tropism reduced to null in the absence of the multispecific binding molecule; and

- modified tropism upon combination with the multispecific binding molecule compared to a reference AAV capsid, wherein the reference AAV capsid comprises a reference AAV capsid protein that is identical to the recombinant AAV capsid protein except for the lack of the heterologous epitope.

The present invention further provides a composition of the present invention for use in a method of therapy or diagnosis in vivo by administering the nucleotide of interest to a target cell expressing a cell surface protein comprising contacting the target cell with the composition, wherein the multispecific binding molecule comprises a retargeting ligand that binds to the cell surface protein.

The present invention also provides a method for administering in vitro or ex vivo a nucleotide of interest to a target cell expressing a cell surface protein comprising contacting the target cell with the composition of the present invention, wherein the multispecific binding molecule comprises a retargeting ligand that binds to the cell surface protein.

Described herein are recombinant AAV capsid proteins comprising an epitope that is heterologous to the capsid protein, wherein the epitope or portion thereof specifically binds to an antibody paratope and wherein the recombinant AAV capsid protein or an AAV capsid comprising the recombinant AAV capsid protein has a reduced to abolished natural tropism, for example, in the absence of a multispecific, optionally bispecific, binding moiety.

In some embodiments, the heterologous epitope is inserted (presented) by the recombinant AAV capsid protein such that the insertion and/or presentation of the heterologous epitope reduces or abolishes the (natural) tropism of the AAV capsid as compared to a reference AAV capsid lacking the heterologous epitope, e.g., the AAV capsid comprises a mutation comprising an insertion of the epitope at the amino acid position and/or a substitution of an amino acid for the epitope at the amino acid position, wherein the mutation reduces or abolishes the (natural) tropism of the AAV capsid protein, e.g., in the absence of a multispecific, optionally bispecific, binding moiety. In some embodiments, the heterologous epitope is inserted (presented) by the AAV capsid protein such that the insertion and/or presentation partially reduces the (natural) tropism of the recombinant AAV capsid as compared to a reference AAV capsid lacking the heterologous epitope, e.g., in the absence of a multispecific, optionally bispecific binding moiety, and the AAV capsid further comprises an additional mutation (e.g., a substitution, deletion, insertion other than insertion of a heterologous epitope) that further reduces and/or abolishes the (natural) tropism of the recombinant AAV capsid or recombinant AAV vectors comprising the same, e.g., in the absence of a multispecific, optionally bispecific binding moiety, as compared to a reference AAV capsid lacking the mutation.

Generally, the recombinant AAV capsid proteins described herein can be derived from an AAV capsid gene, for example, are encoded by an AAV capsid gene modified to express the epitope and/or is a genetically modified AAV. In some embodiments, a recombinant AAV capsid protein described herein is derived from an AAV capsid gene, for example, is encoded by a capsid gene modified to express the epitope and/or is a genetically modified adeno-associated virus (AAV) capsid protein of an AAV serotype that infects primates, wherein optionally the AAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. In some embodiments, the recombinant AAV capsid protein is derived from an AAV2, AAV6, AAV8, or AAV9 capsid gene, for example, is a genetically modified AAV2 capsid protein, a genetically modified AAV6 capsid protein, a genetically modified AAV8 capsid protein, or a genetically modified AAV9 capsid protein. In some embodiments, the recombinant AAV capsid protein is derived from an AAV2 capsid gene, for example, is encoded by an AAV2 capsid gene modified to express the epitope and/or is a genetically modified AAV2 VP1, VP2, and/or VP3 capsid protein, for which the amino acid sequence of a wild-type AAV2 VP1 protein is respectively set forth as SEQ ID NO:1. In some embodiments, the recombinant AAV capsid protein is derived from an AAV6 capsid gene, for example, is encoded by an AAV6 capsid gene modified to express the epitope and/or is a genetically modified AAV6 VP1, VP2, and/or VP3 capsid protein, for which the wild-type AAV6 VP1 amino acid sequences are set forth as SEQ ID NO:3. In some embodiments, the recombinant AAV capsid protein is derived from an AAV8 capsid gene, for example, is encoded by an AAV8 capsid gene modified to express the epitope and/or is a genetically modified AAV8 VP1, VP2, and/or VP3 capsid protein, for which the amino acid sequence of a wild-type AAV VP1 protein is respectively set forth as SEQ ID NO:21. In some embodiments, the recombinant AAV capsid protein is derived from an AAV9 capsid gene, for example, is encoded by an AAV9 capsid gene modified to express the epitope and/or is a genetically modified AAV9 VP1, VP2, or VP3 capsid protein, for which the wild-type amino acid sequence of AAV9 VP1 is respectively set forth as SEQ ID NO:5. In some embodiments, the recombinant AAV capsid protein is derived from an AAV2 capsid gene, e.g., is encoded by an AAV2 capsid gene modified to express the epitope and/or is a genetically modified VP1, VP2, and/or VP3 capsid protein of AAV2. In some embodiments, the recombinant AAV capsid protein is derived from an AAV6 capsid gene, e.g., is encoded by an AAV6 capsid gene modified to express the epitope and/or is a genetically modified VP1, VP2, and/or VP3 capsid protein of AAV6. In some embodiments, the recombinant AAV capsid protein is derived from an AAV8 capsid gene, e.g., is encoded by an AAV8 capsid gene modified to express the epitope and/or is a genetically modified VP1, VP2, and/or VP3 capsid protein of AAV8. In some embodiments, the recombinant AAV capsid protein is derived from an AAV9 capsid gene, e.g., is encoded by an AAV9 capsid gene modified to express the epitope and/or is a genetically modified VP1, VP2, and/or VP3 capsid protein of AAV9.

In some embodiments, the recombinant AAV capsid protein is derived from, for example, is encoded by, a chimeric AAV capsid gene modified to express the epitope, wherein the chimeric AAV capsid gene comprises a plurality of nucleic acid sequences, wherein each of the plurality of nucleic acid sequences encodes a portion of an AAV capsid protein from a different AAV serotype, and wherein the plurality of nucleic acid sequences together encode a chimeric AAV capsid protein. In some embodiments, the recombinant AAV capsid protein is derived from a chimeric AAV2 capsid gene. In some embodiments, the recombinant AAV capsid protein is derived from a chimeric AAV6 capsid gene. In some embodiments, the AAV capsid protein is derived from a chimeric AAV8 capsid gene. In some embodiments, the recombinant AAV capsid protein is derived from a chimeric AAV9 capsid gene.

Generally, a recombinant AAV capsid protein as described herein comprises a heterologous epitope inserted into and/or presented by the recombinant AAV capsid protein such that the heterologous epitope itself reduces and/or abolishes the natural tropism of the recombinant AAV capsid protein or the capsid comprising the same, as compared to a reference AAV capsid protein lacking the heterologous epitope or an AAV capsid comprising the reference AAV capsid protein, respectively. In some embodiments, the heterologous epitope is inserted into (presented in) a region of the AAV capsid protein involved with the natural tropism of the reference wild-type AAV capsid protein, e.g., a region of the AAV capsid protein involved in cell targeting. In some embodiments, the heterologous epitope is inserted into and/or presented via a knob domain of an Ad fiber protein. In some embodiments, the heterologous epitope is inserted into and/or presented via the HI loop of an Ad fiber protein. In some embodiments, the heterologous epitope is inserted into and/or presented via the heparin binding site of an AAV capsid protein. In some embodiments, the heterologous epitope is inserted into and/or presented via the heparin binding site of an AAV2 capsid protein. In some embodiments, the heterologous epitope is inserted into and/or presented via the heparin binding site of an AAV6 capsid protein. In some embodiments, the heterologous epitope is inserted into and/or presented via the heparin binding site of an AAV8 capsid protein. In some embodiments, the heterologous epitope is inserted and/or presented at the heparin binding site of an AAV9 capsid protein. In some embodiments, (i) the AAV capsid protein is derived from an AAV2 capsid gene and the epitope is inserted after and/or replaces an amino acid at position I-453 or I-587 of the AAV2 VP1 capsid protein and/or amino acids at the corresponding positions of the AAV2 VP2 and/or VP3 capsid proteins; (ii) the AAV capsid protein is derived from an AAV6 capsid gene and the epitope is inserted after and/or replaces an amino acid at position 1-585 of the AAV6 VP1 capsid protein and/or amino acids at the corresponding positions of the AAV6 VP2 and/or VP3 capsid proteins; (iii) the AAV capsid is derived from an AAV8 capsid gene and the epitope is inserted after and/or replaces an amino acid at position 1-590 of the AAV8 VP1 capsid protein and/or amino acids at corresponding positions in the AAV8 VP2 and/or VP3 capsid proteins, or (iv) the AAV capsid protein is derived from an AAV9 capsid gene and the epitope is inserted after and/or replaces an amino acid at position I-453 or I-589 of the AAV9 VP1 capsid protein and/or amino acids at corresponding positions in the AAV9 VP2 and/or VP3 capsid proteins. In some embodiments, the heterologous epitope is inserted immediately after (e.g., fused to the C-terminus of) an amino acid selected from the group consisting of G-453 of the AAV2 capsid protein VP1 (or the corresponding positions of the VP2 and/or VP3 capsid proteins encoded from the same capsid gene, or the corresponding amino acids of the VP1, VP2, and/or VP3 capsid proteins of a different AAV that infects humans, e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9), N587 of the AAV2 VP1 capsid protein (or the corresponding positions of the VP2 and/or VP3 capsid proteins encoded from the same capsid gene, or the corresponding amino acids of the VP1, VP2, and/or VP3 capsid proteins of a different AAV that infects humans, e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9), N588 of the AAV2 VP1 capsid protein (or the corresponding positions of the VP2 and/or VP3 capsid proteins encoded from the same capsid gene, or the corresponding amino acids of the VP1, VP2, and/or VP3 capsid proteins of a different AAV that infects humans, e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9), N589 of the AAV2 VP1 capsid protein (or the corresponding positions of the VP2 and/or VP3 capsid proteins encoded from the same capsid gene, or the corresponding amino acids of the VP1, VP2, and/or VP3 capsid proteins of a different AAV that infects humans, e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, A VP1, VP2, and/or VP3 capsid proteins from a different AAV that infects humans, e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9), Q585 of the VP1 capsid protein of AAV6 (or the corresponding positions of the VP2 and/or VP3 capsid proteins encoded from the same capsid gene, or the corresponding amino acids of the VP1, VP2, and/or VP3 capsid proteins from a different AAV that infects humans, e.g., AAV1, AAV2, AAV3, A<a>V4, AAV5, AAV7, AAV8, and AAV9), N-590 of the VP1 capsid protein of AAV8 (or the corresponding positions of the VP2 and/or VP3 capsid proteins encoded from the same capsid gene, or the corresponding amino acids of the VP1, VP2, and/or VP3 capsid proteins of a different AAV that infects humans, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV9), G453 of the VP1 capsid protein of AAV9 (or the corresponding positions of the VP2 and/or VP3 capsid proteins encoded from the same capsid gene, or the corresponding amino acids of VP1, VP2, and/or VP3 of a different AAV that infects humans, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV8), or A-589 of the VP1 capsid protein of AAV9 (or the corresponding positions of the proteins of the VP2 and/or VP3 capsid proteins encoded from the same capsid gene, or the corresponding amino acids of the VP1, VP2, and/or VP3 capsid proteins of a different AAV that infects humans, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV8). In some embodiments, the heterologous epitope is inserted immediately after (e.g., fused to the C-terminus of) G-453 of the AAV2 VP1 capsid protein (or the corresponding positions of the VP2 and/or VP3 capsid proteins encoded from the same capsid gene, or the corresponding amino acids of the VP1, VP2, and/or VP3 capsid proteins of a different AAV that infects humans, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9). In some embodiments, the heterologous epitope is inserted immediately after (e.g., fused to the C-terminus of) N587 of the AAV2 VP1 capsid protein (or the corresponding positions of the VP2 and/or VP3 capsid proteins encoded from the same capsid gene, or the corresponding amino acids of the VP1, VP2, and/or P3 capsid proteins of a different AAV that infects humans, e.g., AAV1, AAV3, AAV4, A5, AAV6, AAV7, AAV8, and AAV9). In some embodiments, the heterologous epitope is inserted immediately after (e.g., fused to the C-terminus of) Q-585 of the AAV6 VP1 capsid protein (or the corresponding positions of the VP2 and/or VP3 capsid proteins encoded from the same capsid gene, or the corresponding amino acids of the VP1, VP2, and/or VP3 capsid proteins of a different AAV that infects humans, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, and AAV9). In some embodiments, the heterologous epitope is inserted immediately after (e.g., fused to the C-terminus of) N-590 of the AAV8 VP1 capsid protein (or the corresponding positions of the VP2 and/or VP3 capsid proteins encoded from the same capsid gene, or the corresponding amino acids of the VP1, VP2, and/or VP3 capsid proteins of a different AAV that infects humans, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, and AAV9). In some embodiments, the heterologous epitope is inserted immediately after (e.g., fused to the C-terminus of) G453 of the VP1 capsid protein of AAV9 (or the corresponding positions of the VP2 and/or VP3 capsid proteins encoded from the same capsid gene, or the corresponding amino acids of the VP1, VP2, and/or VP3 capsid proteins of a different AAV that infects humans, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV8). In some embodiments, the heterologous epitope is inserted immediately after (e.g., fused to the C-terminus of) A-589 of the AAV9 VP1 capsid protein (or the corresponding positions of the VP2 and/or VP3 capsid proteins encoded from the same capsid gene, or the corresponding amino acids of the VP1, VP2, and/or VP3 capsid proteins of a different AAV that infects humans, e.g., AA<v>1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV8). In some embodiments, the heterologous epitope is inserted and/or presented between amino acids N-587 and R-588 of an AAV2 VP1 capsid protein (or the corresponding positions of VP2 and/or VP3 capsid proteins encoded from the same capsid gene). In some embodiments, a recombinant AAV capsid, AAV vector comprising a recombinant AAV capsid, and/or compositions comprising a recombinant AAV capsid comprise an amino acid sequence set forth as SEQ ID NO:2. In some embodiments, a recombinant AAV capsid, AAV vector comprising a recombinant AAV capsid, and/or compositions comprising a recombinant AAV capsid comprise an amino acid sequence set forth as SEQ ID NO:4. In some embodiments, a recombinant AAV capsid, the AAV vector comprising a recombinant AAV capsid, and/or the compositions comprising a recombinant AAV capsid comprise an amino acid sequence encoded by the nucleic acid sequence set forth as SEQ ID NO:25. In some embodiments, a recombinant AAV capsid, the AAV vector comprising a recombinant AAV capsid, and/or the compositions comprising a recombinant AAV capsid comprise an amino acid sequence encoded by the nucleic acid sequence set forth as SEQ ID NO:26. In some embodiments, a recombinant AAV capsid, the AAV1 vector comprising a recombinant AAV capsid, and/or the compositions comprising a recombinant AAV capsid comprise an amino acid sequence encoded by the nucleic acid sequence set forth as SEQ ID NO:27.

In some embodiments, a recombinant AAV capsid protein as described herein comprises a second and different mutation, in addition to the heterologous epitope. For example, in some embodiments, a recombinant AAV capsid protein as described herein may be a genetically modified AAV2 capsid protein, comprise a heterologous epitope, and may further comprise a mutation, for example, an R-585-A and/or R-588-A mutation. In some embodiments, a recombinant AAV capsid protein is derived from an AAV2 capsid gene, e.g., is a genetically modified AAV2 VP1 capsid protein, comprises a heterologous epitope inserted immediately after (e.g., fused to the C-terminus of) G-453 of the AAV2 VP1 protein, and further comprises a mutation selected from the group consisting of R-585A and/or R-5889A. In some embodiments, a recombinant AAV capsid protein is derived from an AAV2 capsid gene, e.g., is a genetically modified AAV2 VP1 capsid protein, comprises a heterologous epitope inserted immediately after (e.g., fused to the C-terminus of) N587 of the AAV2 VP1 protein, and further comprises a mutation selected from the group consisting of R-585A and/or R-588A. In some embodiments, a recombinant AAV capsid protein is derived from an AAV9 capsid gene, e.g., is a genetically modified AAV9 VP1 capsid protein, comprises a heterologous epitope inserted immediately after (e.g., fused to the C-terminus of) G453 of the AAV9 VP1 protein, and further comprises a W503A mutation. In some embodiments, a recombinant AAV capsid protein is derived from an AAV9 capsid gene, e.g., is a genetically modified AAV9 VP1 capsid protein, comprises a heterologous epitope inserted immediately after (e.g., fused to the C-terminus of) A589 of the AAV9 VP1 capsid protein, and further comprises a W503A mutation.

Generally, a recombinant AAV capsid protein and/or an AAV vector comprising the recombinant AAV capsid comprises a heterologous epitope that is at least one amino acid in length. In some embodiments, the heterologous epitope can be between about 5 amino acids and about 35 amino acids in length, and forms a binding pair with an antibody paratope, e.g., an immunoglobulin variable domain. In some embodiments, the heterologous epitope comprises at least 10 amino acids in length. In some embodiments, the heterologous epitope comprises an affinity tag. In some aspects, the heterologous epitope and/or affinity tag does not form a binding pair with an immunoglobulin constant domain. In some aspects, the heterologous epitope and/or affinity tag does not form a binding pair with a metal ion, e.g., Ni2+, Co2+, Cu2+, Zn2+, Fe3+, etc. In some embodiments, the heterologous epitope is not a polypeptide selected from the group consisting of streptavidin, Strep II, HA, L14, 4C-RGD, LH, and Protein A. In some embodiments, the affinity tag is selected from the group consisting of FLAG (SEQ ID NO:7), HA (SEQ ID NO:8), and c-myc (EQKLISEEDL; SEQ ID NO:6). In some embodiments, the heterologous epitope comprises c-myc (EQKLISEEDL; SEQ ID NO:6).

In some embodiments, a recombinant AAV capsid protein is a genetically modified AAV2 VP1 capsid protein and comprises a heterologous epitope comprising the sequence EQKLISEEDL (SEQ ID NO:6) inserted immediately after (e.g., fused to the C-terminus of) G-453 of the AAV2 VP1 capsid protein. In some embodiments, a recombinant AAV capsid protein (i) is derived from an AAV2 capsid gene, e.g., is a genetically modified AAV2 VP1 capsid protein, (ii) comprises a heterologous epitope comprising the sequence EQKLISEEDL (SEQ ID NO:6) and is inserted immediately after (e.g., fused to the C-terminus of) G-453 of the AAV2 VP1 capsid protein, and (iii) further comprises a mutation selected from the group consisting of R-585A and/or R-5889-A. In some embodiments, a recombinant AAV capsid protein is a genetically modified AAV2 VP1 capsid protein and comprises a heterologous epitope comprising the sequence EQKLISEEDL (SEQ ID NO:6) inserted immediately after (e.g., fused to the C-terminus of) N587 of the AAV2 VP1 capsid protein. In some embodiments, a recombinant AAV capsid protein (i) is derived from an AAV2 capsid gene, e.g., is a genetically modified AAV2 VP1 capsid protein, (ii) comprises a heterologous epitope comprising the sequence EQKLISEEDL (SEQ ID NO:6) and is inserted immediately after (e.g., fused to the C-terminus of) N587 of the AAV2 VP1 capsid protein, and (iii) further comprises a mutation selected from the group consisting of R585A and/or R588A. In some embodiments, a recombinant AAV capsid protein (i) is derived from an AAV6 capsid gene, e.g., is a genetically modified AAV6 VP1 capsid protein, (ii) comprises a heterologous epitope comprising the sequence EQKLISEEDL (SEQ ID NO:6) and is inserted immediately after (e.g., fused to the C-terminus of) Q-585 of the AAV6 VP1 capsid protein. In some embodiments, a recombinant AAV capsid protein is a genetically modified AAV8 VP1 capsid protein and comprises a heterologous epitope comprising the sequence EQKLISEEDL (SEQ ID NO:6) inserted immediately after (e.g., fused to the C-terminus of) N590 of the AAV8 VP1 capsid protein. In some embodiments, a recombinant AAV capsid protein is a genetically modified AAV9 VP1 capsid protein and comprises a heterologous epitope comprising the sequence EQKLISEEDL (SEQ ID NO:6) inserted immediately after (e.g., fused to the C-terminus of) G453 of the AAV9 VP1 capsid protein. In some embodiments, a recombinant AAV capsid protein (i) is derived from an AAV9 capsid gene, e.g., is a genetically modified AAV9 VP1 capsid protein, (ii) comprises a heterologous epitope comprising the sequence EQKLISEEDL (SEQ ID NO:6) and is inserted immediately after (e.g., fused to the C-terminus of) G453 of the AAV9 VP1 capsid protein, and (iii) further comprises a W503A mutation. In some embodiments, a recombinant AAV capsid protein is a genetically modified AAV9 VP1 capsid protein and comprises a heterologous epitope comprising the sequence EQKLISEEDL (SEQ ID NO:6) inserted immediately after (e.g., fused to the C-terminus of) A589 of the AAV9 VP1 capsid protein. In some embodiments, a recombinant AAV capsid protein (i) is derived from an AAV9 capsid gene, e.g., is a genetically modified AAV9 VP1 capsid protein, (ii) comprises a heterologous epitope comprising the sequence EQKLISEEDL (SEQ ID NO:6) and is inserted immediately after (e.g., fused to the C-terminus of) A589 of the AAV9 VP1 capsid protein, and (iii) further comprises a W503A mutation.

In some embodiments, a recombinant AAV capsid as described herein comprises the amino acid sequence EQKLISEEDL (set forth as SEQ ID NO:6) flanked by and/or operably linked to at least 5 contiguous amino acids of an AAV VP1 capsid protein. In some embodiments, a recombinant AAV capsid as described herein comprises the amino acid sequence EQKLISEEDL (set forth as SEQ ID NO:6) flanked by and/or operably linked to at least 5 contiguous amino acids of an AAV2 VP1 capsid protein. In some embodiments, a recombinant AAV capsid as described herein comprises EQKLISEEDL (set forth as SEQ ID NO:6) inserted at I587 of an AAV2 VP1 capsid protein. In some embodiments, a recombinant AAV capsid as described herein comprises EQKLISEEDL (set forth as SEQ ID NO:6) inserted between N587 and R588 of an AAV2 VP1 capsid protein, e.g., comprising an amino acid sequence set forth as SEQ ID NO:2.

In some embodiments, a recombinant AAV capsid as described herein comprises the amino acid sequence EQKLISEEDL (set forth as SEQ ID NO:6) flanked by and/or operably linked to at least 5 contiguous amino acids of an AAV6 VP1 capsid protein. In some embodiments, a recombinant AAV capsid as described herein comprises EQKLISEEDL (set forth as SEQ ID NO:6) inserted at 1585 of an AAV6 VP1 capsid protein. In some embodiments, a recombinant AAV capsid as described herein comprises EQKLISEEDL (set forth as SEQ ID NO:6) inserted between Q585 and S586 of an AAV6 Vp 1 capsid protein, e.g., comprises an amino acid sequence set forth as SEQ ID NO:4.

In some embodiments, a recombinant AAV capsid as described herein comprises the amino acid sequence EQKLISEEDL (set forth as SEQ ID NO:6) flanked by and/or operably linked to at least 5 contiguous amino acids of an AAV8 VP1 capsid. In some embodiments, a recombinant AAV capsid comprises EQKLISEEDL (set forth as SEQ ID NO:6) inserted at I590 of an AAV8 VP1 capsid protein. In some embodiments, a recombinant AAV capsid comprises EQKLISEEDL (set forth as SEQ ID NO:6) inserted between N590 and T591 of an AAV8 VP1 capsid protein, e.g., comprises an amino acid sequence set forth as SEQ ID NO:25.

In some embodiments, a recombinant AAV capsid as described herein comprises the amino acid sequence EQKLISEEDL (set forth as SEQ ID NO:6) flanked by and/or operably linked to at least 5 contiguous amino acids of an AAV9 VP1 capsid protein. In some embodiments, a recombinant AAV capsid comprises EQKLISEEDL (set forth as SEQ ID NO:6) inserted at I453 of an AAV9 VP1 capsid protein. In some embodiments, a recombinant AAV capsid comprises EQKLISEEDL (set forth as SEQ ID NO:6) inserted between G-453 and S-454 of an AAV9 VP1 capsid protein, e.g., comprising an amino acid sequence set forth as SEQ ID NO:26. In some embodiments, a recombinant AAV capsid comprises EQKLISEEDL (set forth as SEQ ID NO:6) inserted at I589 of an AAV9 VP1 capsid protein. In some embodiments, a recombinant AAV capsid comprises EQKLISEEDL (set forth as SEQ ID NO:6) inserted between A-589 and Q-590 of an AAV9 VP1 capsid protein, e.g., comprising an amino acid sequence set forth as SEQ ID NO:27.

In some embodiments, the heterologous epitope comprises an affinity tag and one or more linkers. In some embodiments, the heterologous epitope comprises an affinity tag flanked by a linker, e.g., the heterologous epitope comprises from N-terminus to C-terminus a first linker, an affinity tag, and a second linker. In some embodiments, the first and second linkers are each independently a polypeptide at least 1 amino acid in length. In some embodiments, a heterologous epitope as described herein, e.g., an affinity tag alone or together with one or more linkers, has a length between about 5 amino acids and 35 amino acids. In some embodiments, the first and second linkers have identical lengths and/or comprise identical amino acid sequences.

Generally, in the absence of an appropriate multispecific, optionally bispecific, binding molecule, a recombinant AAV capsid comprising a recombinant AAV capsid protein as described herein has been reduced to the point of abrogating natural tropism, e.g., has a reduced ability or is incapable of targeting and binding to a reference cell in a naturally permissive manner for transduction as compared to that of a reference AAV capsid, e.g., a capsid comprising a reference AAV capsid protein, e.g., an AAV capsid protein that would be identical to the recombinant AAV capsid protein except for the lack of the heterologous epitope. In some embodiments and in the absence of an appropriate multispecific, optionally bispecific, binding molecule, a recombinant AAV capsid comprising a recombinant AAV capsid protein as described herein exhibits at least a 10% decrease in transduction efficiency compared to a reference AAV capsid. In some embodiments and in the absence of an appropriate multispecific, optionally bispecific, binding molecule, a recombinant AAV capsid comprising a recombinant AAV capsid protein as described herein exhibits at least a 20% decrease in transduction efficiency compared to a reference AAV capsid. In some embodiments and in the absence of an appropriate multispecific, optionally bispecific, binding molecule, a recombinant AAV capsid comprising a recombinant AAV capsid protein as described herein exhibits at least a 30% decrease in transduction efficiency compared to a reference AAV capsid. In some embodiments and in the absence of an appropriate multispecific, optionally bispecific, binding molecule, a recombinant AAV capsid comprising a recombinant AAV capsid protein as described herein exhibits at least a 40% decrease in transduction efficiency compared to a reference AAV capsid. In some embodiments and in the absence of an appropriate multispecific, optionally bispecific, binding molecule, a recombinant AAV capsid comprising a recombinant AAV capsid protein as described herein exhibits at least a 50% decrease in transduction efficiency compared to a reference AAV capsid. In some embodiments and in the absence of an appropriate multispecific, optionally bispecific, binding molecule, a recombinant AAV capsid comprising a recombinant AAV capsid protein as described herein exhibits at least a 60% decrease in transduction efficiency compared to a reference AAV capsid. In some embodiments and in the absence of an appropriate multispecific, optionally bispecific, binding molecule, a recombinant AAV capsid comprising a recombinant AAV capsid protein as described herein exhibits at least a 70% decrease in transduction efficiency compared to a reference AAV capsid. In some embodiments and in the absence of an appropriate multispecific, optionally bispecific, binding molecule, a recombinant AAV capsid comprising a recombinant AAV capsid protein as described herein exhibits at least a 75% decrease in transduction efficiency compared to a reference AAV capsid. In some embodiments and in the absence of an appropriate multispecific, optionally bispecific, binding molecule, a recombinant AAV capsid comprising a recombinant AAV capsid protein as described herein exhibits at least an 80% decrease in transduction efficiency compared to a reference AAV capsid. In some embodiments and in the absence of an appropriate multispecific, optionally bispecific, binding molecule, a recombinant AAV capsid comprising a recombinant AAV capsid protein as described herein exhibits at least an 85% decrease in transduction efficiency compared to a reference AAV capsid. In some embodiments and in the absence of an appropriate multispecific, optionally bispecific, binding molecule, a recombinant AAV capsid comprising a recombinant AAV capsid protein as described herein exhibits at least a 90% decrease in transduction efficiency compared to a reference AAV capsid. In some embodiments and in the absence of an appropriate multispecific, optionally bispecific, binding molecule, a recombinant AAV capsid comprising a recombinant AAV capsid protein as described herein exhibits at least a 95% decrease in transduction efficiency compared to a reference AAV1 capsid. In some embodiments and in the absence of an appropriate multispecific, optionally bispecific, binding molecule, a recombinant AAV capsid comprising a recombinant AAV capsid protein as described herein exhibits at least a 99% decrease in transduction efficiency compared to a reference AAV capsid. In some embodiments and in the absence of an appropriate multispecific, optionally bispecific, binding molecule, transduction of a control cell by a recombinant AAV capsid comprising a recombinant AAV capsid protein as described herein is abolished, e.g., is undetectable.

In some embodiments, an AAV capsid comprising a recombinant AAV capsid protein as described herein is a mosaic capsid, e.g., it comprises a recombinant AAV capsid protein comprising a heterologous epitope and a reference AAV capsid protein that does not comprise the heterologous epitope in a certain ratio. In some embodiments, a reference AAV capsid protein is a wild-type reference AAV capsid protein thereby comprising an amino acid sequence of a wild-type AAV capsid protein having the same serotype as the recombinant AAV capsid protein. In some embodiments, a reference AAV capsid protein is a control reference AAV capsid protein in that it comprises an amino acid sequence of the recombinant reference AAV capsid protein except that the control reference AAV capsid protein lacks the heterologous epitope. In some embodiments, a reference AAV capsid protein is a mutated wild-type reference protein in that it comprises an amino acid sequence substantially identical to that of a wild-type AAV capsid protein having the same serotype as the recombinant AAV capsid protein except for a mutation, (e.g., an amino acid sequence insertion, chimerization, etc.) that reduces the tropism of the wild-type AAV capsid protein. In some embodiments, a composition described herein comprises, or a method described herein combines, a recombinant AAV capsid protein and a reference capsid protein in a ratio ranging from 1:1 to 1:15. In some embodiments, the ratio is 1:2. In some embodiments, the ratio is 1:3. In some embodiments, the ratio is 1:4. In some embodiments, the ratio is 1:5. In some embodiments, the ratio is 1:6. In some embodiments, the ratio is 1:7. In some embodiments, the ratio is 1:8. In some embodiments, the ratio is 1:9. In some embodiments, the ratio is 1:10. In some embodiments, the ratio is 1:11. In some embodiments, the ratio is 1:12. In some embodiments, the ratio is 1:13. In some embodiments, the ratio is 1:14. In some embodiments, the ratio is 1:15.

Also disclosed herein are nucleic acids encoding a recombinant AAV capsid protein described herein, compositions comprising such nucleic acids (e.g., which can be used in methods for producing a recombinant AAV capsid) and/or recombinant AAV capsid proteins (e.g., compositions consisting substantially of a recombinant AAV capsid protein, compositions comprising only AAV vectors encapsulated by a capsid comprising an AAV capsid protein described herein, compositions comprising such AAV vectors and a multispecific, optionally bispecific, binding molecule (e.g., in certain ratios of AAV vector to multispecific, optionally bispecific, binding molecule (molecule:molecule), compositions comprising such AAV vectors, targeting moieties, and a pharmaceutically acceptable carrier, etc.). In some embodiments, a nucleic acid as described herein comprises a nucleotide sequence encoding the amino acid sequence of EQKLISEEDL (SEQ ID NO:6) and a nucleotide sequence encoding at least 5 contiguous amino acids of an adeno-associated virus capsid protein.

In some embodiments, a nucleic acid as described herein comprises a nucleotide sequence encoding the amino acid sequence EQKLISEEDL (set forth as SEQ ID NO:6) flanked by and/or operably linked to at least 5 contiguous amino acids of an AAV2 VP1 capsid protein. In some embodiments, a nucleic acid as described herein comprises a nucleotide sequence encoding EQKLISEEDL (set forth as SEQ ID NO:6) inserted at I587 of an AAV2 VP1 capsid protein. In some embodiments, a nucleic acid as described herein comprises a nucleotide sequence encoding EQKLISEEDL (set forth as SEQ ID NO:6) inserted between N587 and R588 of an AAV2 VP1 capsid protein, for example, in some embodiments, a nucleic acid as described encodes an amino acid sequence comprising an amino acid sequence set forth as SEQ ID NO:2.

In some embodiments, a nucleic acid as described herein comprises a nucleotide sequence encoding the amino acid sequence EQKLISEEDL (set forth as SEQ ID NO:6) flanked by and/or operably linked to at least 5 contiguous amino acids of an AAV6 VP1 capsid protein. In some embodiments, a nucleic acid as described herein comprises a nucleotide sequence encoding EQKLISEEDL (set forth as SEQ ID NO:6) inserted at I-585 of an AAV6 VP1 capsid protein. In some embodiments, a nucleic acid as described herein comprises a nucleotide sequence encoding EQKLISEEDL (set forth as SEQ ID NO:6) inserted between Q-585 and S-586 of an AAV6 VP1 capsid protein, for example, in some embodiments, a nucleic acid as described encodes an amino acid sequence comprising an amino acid sequence set forth as SEQ ID NO:4.

In some embodiments, a nucleic acid as described herein comprises a nucleotide sequence encoding the amino acid sequence EQKLISEEDL (set forth as SEQ ID NO:6) flanked by and/or operably linked to at least 5 contiguous amino acids of an AAV8 VP1 capsid protein. In some embodiments, a nucleic acid as described herein comprises a nucleotide sequence encoding EQKLISEEDL (set forth as SEQ ID NO:6) inserted at I-590 of an AAV8 VP1 capsid protein. In some embodiments, a nucleic acid as described herein comprises a nucleotide sequence encoding EQKLISEEDL (set forth as SEQ ID NO:6) inserted between N-590 and T-591 of an AAV8 VP1 capsid protein, for example, in some embodiments, a nucleic acid as described encodes an amino acid sequence comprising an amino acid sequence set forth as SEQ ID NO:25.

In some embodiments, a nucleic acid as described herein comprises a nucleotide sequence encoding the amino acid sequence EQKLISEEDL (set forth as SEQ ID NO:6) flanked by and/or operably linked to at least 5 contiguous amino acids of an AAV9 VP1 capsid protein. In some embodiments, a nucleic acid as described herein comprises a nucleotide sequence encoding EQKLISEEDL (set forth as SEQ ID NO:6) inserted at I453 of an AAV9 VP1 capsid protein. In some embodiments, a nucleic acid as described herein comprises a nucleotide sequence encoding EQKLISEEDL (set forth as SEQ ID NO:6) inserted between G453 and S454 of an AAV9 VP1 capsid protein, for example, in some embodiments, a nucleic acid as described encodes an amino acid sequence comprising an amino acid sequence set forth as SEQ ID NO:26. In some embodiments, a nucleic acid as described herein comprises a nucleotide sequence encoding EQKLISEEDL (set forth as SEQ ID NO:6) inserted at I589 of an AAV9 VP1 capsid protein. In some embodiments, a nucleic acid as described herein comprises a nucleotide sequence encoding EQKLISEEDL (set forth as SEQ ID NO:6) inserted between A589 and Q590 of an AAV9 VP1 capsid protein, for example, in some embodiments, a nucleic acid as described encodes an amino acid sequence comprising an amino acid sequence set forth as SEQ ID NO:27.

Generally, recombinant AAV vectors as described herein comprise an AAV capsid comprising a recombinant AAV capsid protein as described herein, wherein the AAV capsid encapsulates a nucleotide of interest. In some embodiments, the nucleotide of interest is under the control of a promoter selected from the group consisting of a viral promoter, a bacterial promoter, a mammalian promoter, an avian promoter, a fish promoter, an insect promoter, and any combination thereof. In some embodiments, the nucleotide of interest is under the control of a non-human promoter. In some embodiments, the promoter is a cytomegalovirus (CMV) promoter. In some embodiments, the promoter is an EF1a promoter.

Generally, a nucleotide of interest may be one or more genes, which may encode a detectable marker, e.g., reporter, or a therapeutic polypeptide. In some embodiments, the nucleotide of interest is a reporter gene. In some embodiments, the nucleotide of interest is a reporter gene encoding a detectable marker selected from the group consisting of green fluorescent protein, luciferase, p-galactosidase, etc. In some embodiments, the detectable marker is green fluorescent protein. In other embodiments, the nucleotide of interest is selected from the group consisting of a suicide gene, a nucleotide encoding an antibody or fragment thereof, a nucleotide encoding a CRISPR/Cas system or portion(s) thereof, a nucleotide encoding antisense RNA, a nucleotide encoding siRNA, a secreted enzyme, etc. In one embodiment, the nucleotide of interest encodes a multi-domain therapeutic compound, e.g., a protein comprising at least two domains that provide two distinct functions.

The compositions described herein generally comprise an AAV vector comprising a recombinant AAV capsid protein as described herein, e.g., comprising a capsid comprising the recombinant AAV capsid protein, wherein the capsid encapsulates a nucleotide of interest. In some embodiments, a composition described herein comprises (1) an AAV vector having a capsid comprising a recombinant AAV capsid protein genetically modified to comprise a heterologous epitope, (2) a multispecific, optionally bispecific, binding molecule comprising (i) an antibody paratope that specifically binds to the epitope and (ii) a retargeting ligand that specifically binds to a receptor, and optionally (3) a pharmaceutically acceptable carrier.

An antibody paratope as described herein generally comprises at least one complementarity determining region (CDR) that specifically recognizes the heterologous epitope, e.g., a CDR3 region of a heavy and/or light chain variable domain. In some embodiments, a multispecific, optionally bispecific, binding molecule comprises an antibody (or portion thereof) comprising the antibody paratope that specifically binds to the heterologous epitope. For example, a multispecific, optionally bispecific, binding molecule may comprise a single-domain heavy chain variable region or a single-domain light chain variable region, wherein the single-domain heavy chain variable region or the single-domain light chain variable region comprises an antibody paratope that specifically binds to the heterologous epitope. In some embodiments, a multispecific, optionally bispecific binding molecule may comprise an Fv region, e.g., a multispecific, optionally bispecific binding molecule may comprise an scFv, which comprises an antibody paratope that specifically binds to the heterologous epitope. In some embodiments, the multispecific, optionally bispecific binding molecule comprises an antibody (or portion thereof) comprising an antibody paratope that specifically binds to the heterologous epitope, wherein the antibody (or portion thereof) further comprises one or more antibody constant domains (e.g., a heavy chain constant domain (e.g., CH1, hinge, CH2, CH3, CH4, etc.) and/or a light chain constant domain (e.g., CL), wherein one or more antibody constant domains do not bind to the heterologous epitope.

A multispecific, optionally bispecific, binding molecule as described herein further comprises a retargeting ligand, in addition to a paratope (e.g., an antibody or portion thereof comprising the paratope) that specifically binds to the heterologous epitope inserted into/presented by a recombinant AAV capsid protein. In some embodiments, the retargeting ligand specifically binds to a receptor on the surface of a bead (e.g., for isolation and/or purification of a recombinant AAV capsid protein as described herein). In some embodiments, the retargeting ligand specifically binds to a cell surface protein, e.g., a receptor, cell surface marker, etc., expressed on the surface of a mammalian eukaryotic (e.g., human) cell, e.g., a target cell. In some embodiments, a retargeting ligand binds to a (human) liver cell, a (human) brain cell, a (human) T cell, a (human) kidney cell, an (human) intestinal cell, a (human) pancreas cell, a (human) cancer cell, and/or a (human) cell infected with a heterologous pathogen. In some embodiments, a retargeting ligand binds to a (human) liver cell-specific marker, a (human) brain cell-specific marker, a (human) T cell-specific marker, a (human) kidney cell-specific marker, an (human) intestinal cell-specific marker, a (human) pancreas cell-specific marker, a (human) tumor cell-specific marker, and/or a pathogen epitope.

In some embodiments, the retargeting ligand binds to a receptor expressed by a liver cell (human), e.g., an asialoglycoprotein receptor, e.g., hASGR1. In some embodiments, the retargeting ligand binds to a receptor expressed by a neuronal cell (human), e.g., GABA, transferrin, etc. In some embodiments, the retargeting ligand binds to a receptor expressed by a T cell (human), e.g., CD3, e.g., CD3e. In some embodiments, the retargeting ligand binds to a receptor expressed by a hematopoietic stem cell (human), e.g., CD34. In some embodiments, the retargeting ligand binds to a receptor expressed by a kidney cell (human). In some embodiments, the retargeting ligand binds to a receptor expressed by a muscle cell (human), e.g., an integrin. In some embodiments, the retargeting ligand binds to a receptor expressed by a (human) cancer cell, e.g., a tumor-associated antigen, e.g., adipophilin, AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein (AFP), ARTC1, B-RAF, BAGE-1, BCLX(L), BCRABL b3a2 fusion protein, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen (CEA), CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin D1, Cyclin-A1, dek-can fusion protein, DKK1, EFTUD2, Elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, antígeno tumoral epitelial (ETA), proteína de fusión de ETV6-AML1, EZH2, E6, E7, FGF5, FLT3-ITD, FN1, G250/MN/CAIX, GAGE-1,2,8, GAGE-3,4,5,6,7, GAS7, glipicano-3, GnTV, gp100/Pme117, GPNMB, HAUS3, Hepsina, HER-2/neu, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, IDOl, IGF2B3, IL13Ralfa2, Carboxil esterasa intestinal, K-ras, Calicreína 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1 (also known as CCDC110), LAGE-1, LDLR-fucosyltransferaseAS fusion protein, Lengsin, M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, melic enzyme, mammaglobin-A, MART2, MATN, MCSP, mdm-2, ME1, Melan-A/MART-1, Meloe, Midquina, MMP-2, MMP-7, MUC1, MUC5AC, mucin, MUM-1, MUM-2, MUM-3, Myosin, Myosin class I, N-raw, NA88-A, neo-PAP, NFYC, NY-BR-1, NY-ESO-1/LAGE-2, OA1, OGT, OS-9, P polypeptide, p53, PAP, PAX5, PBF, pml-RARalpha fusion protein, polymorphic epithelial mucin (PEM), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38/NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, secernin 1, RT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1, survivin, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2, Telomerase, TGF-betaRII, TPBG, TRAG-3, Triosephosphate isomerase, TRP-1/gp75, TRP-2, TRP2-INT2, tyrosinase (TYR), VEGF, WT1, XAGE-lb/GAGED2a, Kras, NY-ESO1, MAGE-A3, HPV E2, HPV E6, HPV E7, WT-1 antigen (in lymphoma and other solid tumors), ErbB receptors, Melan A [MART1], gp 100, tyrosinase, TRP-1/gp 75, and TRP-2 (in melanoma); MAGE-1 and MAGE-3 (in bladder, head and neck, and non-small cell carcinoma); HPV EG and E7 proteins (in cervical cancer); Mucin [MUC-1] (in breast, pancreatic, colon, and prostate cancers); prostate-specific antigen [PSA] (in prostate cancer); carcinoembryonic antigen [CEA] (in colon, breast, and gastrointestinal cancers) and shared tumor-specific antigens such as MAGE-2, Ma GE-4, MAGE-6, MAGE-10, MAGE-12, BAGE-1, CAGE-1,2,8, CAGE-3 TO 7, LAGE-1, NY-ESO-1/LAGE-2, NA-88, GnTV, TRP2-INT2, etc. In some embodiments, the retargeting ligand binds to E6 and/or E7. In some embodiments, the retargeting ligand binds to Her2. In some embodiments, the retargeting ligand binds to the human glucagon receptor (hGCGR). In some embodiments, the retargeting ligand binds to human ectonucleoside triphosphate diphosphohydrolase 3 (hENTPD3).

In some embodiments, the paratope (e.g., an antibody or portion thereof) and the retargeting ligand are directly fused to each other. In some embodiments, the paratope (e.g., an antibody or portion thereof) that specifically binds to the heterologous epitope and the retargeting ligand are covalently linked to each other.

In some embodiments, the multispecific binding molecule is a bispecific binding molecule, e.g., an antibody comprising a first and a second antigen binding domain, wherein the first antigen binding domain comprises a paratope that specifically binds to the heterologous epitope inserted into/presented by a recombinant AAV capsid protein and the second antigen binding domain specifically binds to a protein or cell surface marker expressed by a target cell. In some embodiments, the bispecific binding molecule is a bispecific antibody comprising a first and a second antigen binding domain, wherein the first antigen binding domain comprises a paratope that specifically binds to the heterologous epitope inserted into/presented by a recombinant AAV capsid protein and the second antigen binding domain specifically binds to a receptor expressed by a target cell, wherein the first antigen binding domain is operably linked to a first heavy chain region comprising a first CH3 domain, wherein the second antigen binding domain is operably linked to a second heavy chain region comprising a second CH3 domain, wherein the first and second Ig Ch3 domains differ from each other by at least one amino acid, and wherein the at least one amino acid difference reduces the binding of the bispecific antibody to Protein A as compared to a bispecific antibody lacking the amino acid difference. In one embodiment, the first Ig C<h>3 domain binds protein A and the second Ig C<h>3 domain contains a mutation that reduces or abolishes protein A binding such as an H-95-R modification (according to IMGT exon numbering; H435R according to EU numbering). The second Ch3 domain may further comprise a Y-96-F modification (according to IMGT; Y-436-F according to EU). Additional modifications that may be found within the second C<h>3 domain include: D-16-E, L-18-M, N-44-S, K-52-N, V-57-M, and V-82-I (according to IMGT; D-356-E, L-358-M, N-384-S, K-392-N, V-397-M, and V-422-I according to EU) in the case of IgG1 antibodies; N-44-S, K-52-N and V-82-I (IMGT; N-384-S, K-392-N and V-422-I according to EU) in the case of IgG2 antibodies; and Q-15-R, N-44-S, K-52-N, V-57-M, R-6-9K, E-79-Q and V-82-I (according to IMGT; Q-355-R, N-384-S, K-392-N, V-397-M, R-409-K, E-419-Q and V-422-I according to EU) in the case of IgG4 antibodies.

In some embodiments, the multispecific binding molecule is a bispecific binding molecule, e.g., a bispecific antibody comprising a first and a second antigen binding domain, wherein the first antigen binding domain binds to an affinity tag presented by a recombinant AAV capsid protein as described herein, and wherein the second antigen binding domain binds to a receptor expressed on the surface of a target cell. In some embodiments, the multispecific binding molecule is a bispecific binding molecule, e.g., a bispecific antibody comprising a first and a second antigen binding domain, wherein the first antigen binding domain binds to the amino acid sequence EQKLISEEDL (SEQ ID NO:6) presented by a recombinant AAV capsid protein as described herein, and wherein the second antigen binding domain binds to a receptor expressed on the surface of a target cell. In some embodiments, the multispecific binding molecule is a bispecific binding molecule, e.g., a bispecific antibody comprising a first and a second antigen binding domain, wherein the first antigen binding domain binds to the amino acid sequence EQKLISEEDL (SEQ ID NO:6) presented by a recombinant AAV capsid protein as described herein, and wherein the second antigen binding domain binds to hASGR1. In some embodiments, the multispecific binding molecule is a bispecific binding molecule, e.g., a bispecific antibody comprising a first and a second antigen binding domain, wherein the first antigen binding domain binds to the amino acid sequence EQKLISEEDL (SEQ ID NO:6) presented by a recombinant AAV capsid protein as described herein, and wherein the second antigen binding domain binds to a CD3 protein, e.g., CD3<e>. In some embodiments, the multispecific binding molecule is a bispecific binding molecule, e.g., a bispecific antibody comprising a first and a second antigen binding domain, wherein the first antigen binding domain binds to the amino acid sequence EQKLISEEDL (SEQ ID NO:6) presented by a recombinant AAV capsid protein as described herein, and wherein the second antigen binding domain binds to an integrin. In some embodiments, the multispecific binding molecule is a bispecific binding molecule, e.g., a bispecific antibody comprising a first and a second antigen binding domain, wherein the first antigen binding domain binds to the amino acid sequence EQKLISEEDL (SEQ ID NO:6) presented by a recombinant AAV capsid protein as described herein, and wherein the second antigen binding domain binds to a human glucagon receptor, e.g., hGCGR. In some embodiments, the multispecific binding molecule is a bispecific binding molecule, for example, a bispecific antibody comprising a first and a second antigen binding domain, wherein the first antigen binding domain binds to the amino acid sequence EQKLISEEDL (SEQ ID NO:6) presented by a recombinant AAV capsid protein as described herein, and wherein the second antigen binding domain binds to ENTPD3.

Also described herein are methods for producing and using the recombinant AAV capsid proteins, AAV vectors comprising the same, compositions, etc. In some embodiments, a method for redirecting an adeno-associated virus, etc.; delivering diagnostic/therapeutic payload to a target cell, etc. comprises combining a recombinant AAV vector comprising a recombinant AAV capsid protein as described herein, e.g., an AAV vector comprising a capsid comprising a recombinant AAV capsid presenting a heterologous epitope, with a bispecific binding molecule, wherein the bispecific binding molecule comprises (i) an antibody paratope that specifically binds to the epitope and (ii) a redirection ligand that specifically binds to a receptor. Such methods may include as a first step producing a recombinant AAV vector, e.g., culturing a packaging cell under conditions sufficient for AAV vector production, wherein the packaging cell comprises a plasmid encoding the AAV capsid protein comprising the epitope. In delivering diagnostic/therapeutic payload to a target cell, the methods described herein may comprise contacting the target cell with the combination of an AAV vector comprising an AAV capsid comprising a recombinant AAV capsid presenting a heterologous epitope and a multispecific binding molecule, wherein the multispecific binding molecule comprises (i) an antibody paratope that specifically binds to the epitope and (ii) a retargeting ligand that specifically binds to a receptor expressed by the target cell. In some embodiments, the target cell is in vitro. In other embodiments, the present invention also provides a composition of the present invention for use in a method of treatment or diagnosis in vivo, the method comprising administering the nucleotide of interest to a target cell expressing a cell surface protein comprising contacting the target cell with the composition, wherein the multispecific binding molecule comprises a retargeting ligand that binds to the cell surface protein. Thus, in such embodiments, the target cell is in vivo in a subject, e.g., a human.

In some embodiments, a composition described herein comprises, or a method described herein combines, a recombinant AAV vector comprising a nucleotide of interest encapsulated with a capsid comprising a recombinant capsid protein as described herein and a multispecific binding molecule in a molecule:molecule ratio that restores the transduction efficiency of the AAV vector similar to that of a reference AAV vector. In some embodiments, the ratio of recombinant AAV vector to multispecific binding molecule (molecule:molecule) ranges from 1:0.5 to 1:100. In some embodiments, the ratio of recombinant AAV vector to multispecific binding molecule (molecule:molecule) ranges from 1:4 to 1:20. In some embodiments, the ratio of recombinant AAV vector to bispecific binding molecule (molecule:molecule) ranges from 1:8 to 1:15. In some embodiments, the ratio of recombinant AAV vector to multispecific binding molecule (molecule:molecule) is 1:4. In some embodiments, the ratio of recombinant AAV vector to multispecific binding molecule (molecule:molecule) is 1:8. In some embodiments, the ratio of recombinant AAV vector to multispecific binding molecule (molecule:molecule) is 1:15. In some embodiments, the ratio of recombinant AAV vector to multispecific binding molecule (molecule:molecule) is 1:20.

Also described herein are methods for inactivating an AAV capsid and/or producing AAV vectors, which methods generally comprise (a) inserting a nucleic acid encoding a heterologous protein into a nucleic acid sequence encoding an AAV capsid protein to form a nucleotide sequence encoding a genetically modified AAV capsid protein comprising the heterologous protein and/or (b) culturing a packaging cell under conditions sufficient for the production of AAV vectors, wherein the packaging cell comprises the nucleotide sequence. In some embodiments, the packaging cell further comprises a helper plasmid and/or a transfer plasmid comprising a nucleotide of interest. In some embodiments, the methods further comprise isolating self-complementary adeno-associated viral vectors from the culture supernatant. In some embodiments, the methods further comprise lysing the packaging cell and isolating single-stranded adeno-associated viral vectors from the cell lysate. In some embodiments, the methods further comprise (a) removing cellular debris, (b) treating the supernatant containing AAV vectors with DNase I and MgCl2, (c) concentrating the AAV vectors, (d) purifying the AAV vectors, and (e) any combination of (a)-(d). Also provided herein are an AAV vector made according to the method described herein, and packaging cells useful for producing an AAV vector as described herein, for example, packaging cells comprising a plasmid encoding a recombinant AAV capsid protein are described.

Brief description of the drawings

The patent or application file contains at least one color drawing. Copies of this patent or patent application publication with color drawings will be provided by the office upon request and payment of the necessary fee.

Figure 1 provides immunofluorescence microscopy images (top panel) or histograms obtained from fluorescence-activated cell sorting (FACS) (bottom panel) assessing green fluorescence (GFP) expression by HepG2 cells incubated with (A) wild-type scAAV2-CMV-eGFP viral vectors only; (B) scAAV2-N587Myc-CMV-hrGFP viral vectors only; scAAV2-N587Myc viral vectors mixed with anti-Myc-ASGR1 bispecific antibodies at the following ratios: (C) 1:0.5, (D) 1:1, (E) 1:2, (F) 1:4, (G) 1:8, (H) 1:15, (I) 1:20, (J) 1:50, or (K) 1:100; (L) or scAAV-N587Myc viral vectors mixed with anti-Myc monospecific antibody at a ratio of 1:8.

Figure 2A provides dot plots obtained from fluorescence-activated cell sorting (FACS) assessing green fluorescence (GFP) expression by 29T3-hASGR1 cells incubated with wild-type scAAV2 viral vectors only (i), scAAV2-N587Myc-CMV-eGFP viral vectors only (ii), scAAV2-N587Myc-CMV-eGFP viral vectors mixed with anti-Myc-ASGR1 bispecific antibodies at the following ratios: 1:0.5 (iii), 1:1 (iv), 1:2 (v), 1:4 (vi), 1:8 (vii), 1:15 (viii), 1:20 (ix), or 1:100 (x), or scAAV2-N587Myc-CMV-eGFP viral vectors mixed with anti-Myc-ASGR1 bispecific antibodies. irrelevant bispecific anti-Myc-GCGR at a ratio of 1:8 (xii). GFP expression by 29T3 cells incubated with scAAV-N587Myc viral vectors mixed with anti-Myc-ASGR1 bispecific antibodies at a ratio of 1:8 is also shown (xi). For each experiment, 2x105 cells and 5x109 viral vectors were used.

Figure 2B provides histograms obtained from fluorescence-activated cell sorting (FACS) assessing green fluorescence (GFP) expression by 29T3-hASGR1 cells incubated with unmodified AAV9-CAGG-GFP viral vectors only (i), AAV9-A589Myc-CAGG-eGFP viral vectors only (ii), AAV9-A589Myc-CAGG-eGFP viral vectors mixed with anti-Myc-ASGR1 bispecific antibodies at the following ratios: 1:1 (iii), 1:2 (iv), 1:4 (v), 1:8 (vi), 1:20 (vii), 1:50 (viii), or 1:100 (ix). For each experiment, 2x105 cells and 1x1010 viral vectors (titrated by qPCR) were used.

Figure 3 provides dot plots obtained from fluorescence-activated cell sorting (FACS) assessing green fluorescence (GFP) expression by 29T3-hASGR1 cells preincubated with anti-ASGR1 bivalent antibody at a concentration of 0 nM (C), 50 nM (D), 10 nM (E), 2 nM (F), 0.4 nM (G), 0.08 nM (H), 0.016 nM (I), or 0.0032 nM (J) and subsequently infected with scAAV2-N587Myc-CMV-eGFP viral vectors mixed with anti-Myc-ASGR1 bispecific antibodies at a ratio of 1:8 (L). 293T3-hASGR1 cells incubated with wild-type scAAV viral vectors only (A) or scAAV2-N587Myc-CMV-eGFP viral vectors only (B) serve as controls. For each experiment, 2x105 cells and 5x109 viral vectors (titrated by qPCR) were used.

Figure 4 provides immunofluorescence microscopy images assessing green fluorescence (GFP) expression by 293T-hASGR1 cells incubated with wild-type scAAV viral vectors alone (A), scAAV2-N587Myc-CMV-eGFP viral vectors alone (B), or incubated sequentially with 1x109 (C), 2x109 (D), 4x109 (E), 8x109 (F), 2x1010 (G), 1x1011 (H), 1x1012 (I) anti-Myc-ASGR1 bispecific antibodies followed by 1x109 scAAV2-N587Myc-CMV-eGFP viral vectors. Also shown are Immunofluorescence microscopy images of 293T cells sequentially incubated with 1x1011 anti-myc-ASGR1 antibody molecules followed by 1x109 scAAV2-N587Myc-CMV-eGFP viral vectors (J), and 293T-hASGR1 cells sequentially incubated with 1x1011 irrelevant bispecific anti-Myc-GCGR antibody molecules followed by 1x109 scAAV2-N587Myc-CMV-eGFP viral vectors (K).

Figure 5 provides dot plots obtained from fluorescence-activated cell sorting (FACS) assessing green fluorescence (GFP) expression by 29T3-hASGR1 cells incubated with wild-type ssAAV viral vectors only (A), ssAAV2-N587Myc-CMV-hrGFP viral vectors only (B), ssAAV2-N587Myc-CMV-hrGFP viral vectors mixed with anti-Myc-ASGR1 bispecific antibodies at the following ratios: 1:1 (C), 1:2 (D), 1:4 (E), 1:8 (F), 1:20 (G), 1:100 (H), 1:1000 (I), or ssAAV-N587Myc viral vectors mixed with irrelevant bispecific anti-Myc-GCGR antibody at 1:1 (C). a ratio of 1:8 (K). GFP expression by 29T3 cells incubated with ssAAV-N587Myc viral vectors mixed with anti-Myc-ASGR1 bispecific antibodies at a ratio of 1:8 is also shown (J). For each experiment, 2x105 cells and 5x109 viral vectors were used.

Figure 6 provides dot plots obtained from fluorescence-activated cell sorting (FACS) assessing green fluorescence (GFP) expression by 29T3-hGCGR cells incubated with wild-type scAAV viral vectors only (A), scAAV2-N587Myc-CMV-eGFP viral vectors only (B), scAAV2-N587Myc-CMV-eGFP viral vectors mixed with bispecific anti-Myc-GCGR antibodies at the following ratios: 1:0.5 (C), 1:1 (D), 1:2 (E), 1:4 (F), 1:8 (G), 1:15 (H), 1:20 (I), 1:50 (J), or 1:100 (K), or scAAV2-N587Myc-CMV-eGFP viral vectors mixed with a irrelevant monospecific anti-Myc antibody (Regeneron Pharmaceuticals, Tarrytown, New York) at a ratio of 1:8 (L). For each experiment, 2x105 cells and 5x109 viral vectors were used.

Figure 7 provides dot plots obtained from fluorescence-activated cell sorting (FACS) assessing green fluorescence (GFP) expression by Jurkat cells alone (A) or Jurkat cells incubated with wild-type scAAV6-EF1-eGFP viral vectors only (B), AAV6-Q585Myc-EFla-eGFP viral vectors only (C), AAV6-Q585Myc-EF1a-eGFP viral vectors mixed with anti-Myc-CD3 bispecific antibodies at the following ratios: 1:1 (D), 1:5 (E), 1:10 (F), 1:100 or (G), 1:1000 (H). For each experiment, 2x105 cells and 1x109 viral vectors were used.

Figure 8A provides immunofluorescence microscopy images of the liver. Figure 8B provides immunofluorescence microscopy images of the spleen. Figure 8C provides immunofluorescence microscopy images of the kidney. All samples are taken from C57BL/6 mice transgenically modified to express human ASGR1 via liver cells (i-iv) or wild-type C57BL/6 mice (v-viii) ten days after intravenous injection with 1x1011 wild-type scAAV2-CMV-eGFP (i, v), saline (ii, vi), 1x1011 scAAV2-N587myc-CMV-eGFP viral vectors alone (iii, vii), or scAAV2-N587myc-CMV-eGFP viral vectors with anti-myc-ASGR1 bispecific antibody (iv, viii).

Figure 9 provides immunofluorescence microscopy images of liver samples taken from C57BL/6 mice transgenically modified to express human ASGR1 in liver cells (D-F, J-L, P-R) or wild-type C57BL/6 mice (A-C, G-I, M-O) four weeks after intravenous injection with 2.18x1011 wild-type ssAAV2-CAGG-eGFP (B, C, E, F), saline (A, D), 2.18x1011 ssAAV2-N587myc-CAGG-eGFP viral vectors alone (G-I, J-L), or ssAAV2-N587myc-CAGG-eGFP viral vectors with bispecific anti-myc-ASGR1 antibody (M-O, P-R). Each image represents one mouse.

Figure 10 provides immunofluorescence microscopy images of liver samples taken from C57BL/6 mice transgenically modified to express human ASGR1 via liver cells ten days after intravenous injection with (A) wild-type AAV9, (B) 250 nM NaCl (C) AAV9-A589myc-CAGG-eGFP viral particles together with anti-myc-hCD3 bispecific antibody, or (D) AAV9-A589myc-CAGG-eGFP viral particles together with anti-myc-ASGR1 bispecific antibody.

Figure 11 provides illustrative, non-scale, non-limiting example formats of multispecific binding molecules useful in some embodiments of the invention.

Figure 12 provides dot plots obtained from fluorescence-activated cell sorting (FACS) assessing green fluorescence (GFP) expression by 29T3-hASGR1 cells incubated with (A) wild-type AAV8 viral vectors only, (C) AA8-N590-myc viral vectors only, or pAAV RC8 N590myc viral vectors mixed with anti-hASGR1-IgG4-Fc/anti-myc bispecific molecules at the following ratios (D) 1:1, (E) 1:2, (F) 1:4, (G) 1:8, (H) 1:12, (I) 1:15, (J) 1:50, or (K) 1:100. GFP expression by mock-transfected 29T3-hASGR1 cells is also shown (B). For each experiment, 2x105 cells and 1x109 viral vectors were used.

Figure 13 provides immunofluorescence microscopy images of liver samples taken from C57BL/6 mice transgenically engineered to express human ASGR1 via liver cells ten days after intravenous injection with (A)-(C) wild-type AAV8, (D)-(F) AA8-N590-myc viral vectors and control bispecific binding molecule, or (G)-(I) AAV8 N590myc-CAGG-eGFP viral particles together with anti-hASGR1-IgG4-Fc/anti-myc bispecific binding molecules.

Figure 14 provides dot plots obtained from fluorescence-activated cell sorting (FACS) assessing green fluorescence (GFP) expression by 29T3-hENTPD3 cells incubated with (A) wild-type AAV2 viral vectors only, (B) AAV2-N587Myc-CAGG-eGFP viral vectors only, or AAV2-N587Myc-CAGG-eGFP viral vectors mixed with anti-hENTPD3-IgG4-Fc/anti-myc bispecific molecules at the following ratios (C) 1:1, (D) 1:2, (E) 1:4, (F) 1:8, (G) 1:20, (H) 1:50, (I) 1:100, or (K) 1:200. For each experiment, 2x105 cells and 1x109 viral vectors were used.

Figure 15A provides immunofluorescence microscopy images of liver samples, Figure 15B provides immunofluorescence microscopy images of intestine samples, and Figure 15C provides immunofluorescence microscopy images of pancreas samples. All samples are taken from wild-type C57BL/6 mice ten days after intravenous injection with PBS(15A(i), 15B(i), and 15C(i)), 5x1011 wild-type AAV9(15A(ii), 15B(ii), and15C(ii)), 5x1011 AAV2-N587myc-CAGG-eGFP viral vectors with 1x103 irrelevant bispecific IgG4-Fc/anti-myc binding proteins(15A(iii), 15B(iii), and15C(iii)), or 5x1011 AAV2-N587myc-CAGG-eGFP viral vectors with 1x1013 bispecific hENTPD3-IgG4-Fc/anti-myc binding proteins(15A(iv), 15B(iv) and 15C(iv)).

Detailed description

A common problem with adaptor approaches using either unmodified viral capsids or scaffold-modified viral capsids has been the suboptimal transduction efficiencies of the modified capsids. (Grifman et al. (2001) Mol. Ther. 3:964-75). For example, Curie et al. describe the generation and characterization of a recombinant adenoviral vector containing fibers with an RGD-4C sequence genetically incorporated within the HI loop of the carboxy-terminal knob domain and demonstrate the utility of the HI loop of the fiber knob as an optimal site for incorporation of short peptide ligands. See, e.g., U.S. Patent No. 7,297,542; see also Beatty and Curiel (2012) Av. Cancer. Res. 115:39-67. Similarly, insertion of ligand peptides into AAV capsid proteins resulted in capsids that were capable of presenting the ligand on the capsid surface and mediating transduction through interaction of the ligand with its receptor, thereby redirecting viral tropism through genetic modifications of the capsid (Girodet al.(1999)Nat. Medicina.5(9):1052-6, 1438 (errata included) (1999); Grifmanet al.(2001)Mol. Ther.3(6):964-75; Nicklinet al.(2001)Mol. Ther.4(3):174-81; Shiet al.(2001)Hum Gene Ther.17(3):353-61 (2006); Wuet al.(2000)J. Virol. 74(18):8635-47). In particular, it has been shown that insertion of an integrin-binding Arg-Gly-Asp (RGD) motif into the I-587 insertion site of the AAV capsid protein VP1 allowed AAV viral vectors to transduce cells via avp<1>integrins (Girodet al. (1999) cited above). In contrast, although insertion of the 14 amino acid L14 peptide after amino acid R447 (I-447) led to capsids still being recognized by the conformation-sensitive antibody A20, such recombinant viral vectors were unable to transduce cells expressing the L-14 receptor, (Girodet al.,1999; cf. Wuet al.(2000) (reporting insertion of a hemagglutinin (HA) peptide at position I-447 and successful transduction of cells expressing the HA peptide). Insertion of a Myc epitope between T448 and N449 was recognized by an anti-Myc antibody and was therefore present on the capsid surface, but led to inactivated viral vectors (Grifmanet al.,2001). In contrast, successful retargeting was again reported for insertion of a Myc-specific motif (Grifmanet al.,2001). NGR after N587, but not insertion of c-myc after N587. (Grifman et al.,2001). US Patent No. 9,624,274 describes I-453 of an AAV capsid protein as a suitable insertion site for a heterologous epitope. Although these studies demonstrate successful insertion and presentation of a heterologous peptide, e.g., epitope, by AAV capsid proteins, none of these studies provide any expectation that a multispecific binding molecule, e.g., a bispecific binding molecule such as a bispecific antibody, which specifically binds to the heterologous peptide and a cell surface protein, can redirect modified viral vectors to cells expressing the cell surface protein and restore their transduction efficiencies.

Disclosed herein are recombinant AAV capsid proteins that are modified with a heterologous epitope, which can be used in connection with a multispecific binding molecule comprising a paratope, e.g., an Fv domain, that specifically binds to the epitope and a ligand that binds to a receptor expressed on the surface of a target cell. As shown herein, contacting AAV vectors having capsids formed with the AAV capsid proteins described herein with a multispecific binding molecule in certain ratios restores the transduction efficiency of the AAV capsid to levels comparable to that of the wild-type virus, see, e.g., Example 2. The capsid proteins modified with heterologous epitopes as described herein are derived from adenovirus (Ad) and adeno-associated virus (AAV).

Although the invention has been particularly shown and described with reference to a number of embodiments, it will be understood by those skilled in the art that the various embodiments disclosed herein are not intended to act as limitations on the scope of the claims.

Although any of the methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, some preferred methods and materials are described below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains.

Definitions

Unless otherwise defined, all technical and scientific terms/expressions used herein have the same meaning commonly understood by a person skilled in the art to which the present invention belongs.

The singular forms "a", "an", "the" and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to "a method" includes one or more methods, and/or steps of the type described herein and/or that will become apparent to those skilled in the art after reading this disclosure.

The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains, two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable domain (V<h>) and a heavy chain constant region (C<h>). The heavy chain constant region comprises at least three domains, Cl1, Ch2, Ch3, and optionally CH<4>. Each light chain comprises a light chain variable domain (C<h>) and a light chain constant region (C<l>). The heavy chain and light chain variable domains can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each heavy chain and light chain variable domain comprises three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDr3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2, and HCDR3; light chain CDRs may be abbreviated as LCDR1, LCDR2, and LCDR3. Typical tetrameric antibody structures comprise two identical antigen-binding domains, each formed by association of the Vh and Vl domains, and each of which together with their respective Ch and Cl domains form the Fv region of the antibody. Single-domain antibodies comprise a single antigen-binding domain, for example, a V<h> or a V<l>. The term "antibody" encompasses monoclonal antibodies, multispecific (e.g., bispecific) antibodies, human antibodies, humanized antibodies, antibodies chimeric, single chain Fv (scFv), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fv (sdFv), intrabodies, minibodies, diabodies, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies against antigen-specific TCR) and epitope-binding fragments of any of the foregoing. The terms "antibody" and "antibodies" also refer to covalent diabodies such as those disclosed in US Patent Application Publication 2007/0004909 and Ig-DARTS such as those disclosed in US Patent Application Publication 2009/0060910. Antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

The antigen binding domain of an antibody, e.g., the portion of an antibody that recognizes and binds to the epitope of an antigen, is also referred to as a "paratope." It is a small (5-10 amino acid) region of the Fv region of an antibody, part of the antigen binding fragment (Fab region), and may contain portions of the heavy and/or light chains of the antibody. A paratope specifically binds to an epitope when the paratope binds to the epitope with high affinity. The term "high affinity" antibody refers to an antibody that has a K to its target epitope of about 10.9 M or less (e.g., about 1 x 10.9 M, 1 x 10.10 M, 1 x 10.11 M, or about 1 x 10.12 M). In one embodiment, K is measured by surface plasmon resonance, e.g., BIACORE™; In another embodiment, the Kd is measured by ELISA.

The term "complementarity determining region," or "CDR," includes an amino acid sequence encoded by a nucleic acid sequence of an organism's immunoglobulin genes that normally (i.e., in a wild-type animal) occurs between two framework regions in a variable region of a light chain or a heavy chain of an immunoglobulin molecule (e.g., an antibody or a T-cell receptor). A CDR may be encoded by, for example, a germline sequence or a rearranged or unrearranged sequence, and, for example, by a naive or mature B cell or T cell. A CDR may be somatically mutated (e.g., varied from a sequence encoded in an animal's germline), humanized, and/or modified with amino acid substitutions, additions, or deletions. In some circumstances (e.g., for a CDR3), CDRs may be encoded by two or more sequences (e.g., germline sequences) that are not contiguous (e.g., in an unrearranged nucleic acid sequence) but are contiguous in a B cell nucleic acid sequence, for example, as a result of splicing or joining of the sequences (e.g., V-D-J recombination to form a heavy chain CDR3).

An "epitope" is that part of a macromolecule that is recognized by the immune system, specifically by antibodies, B cells, or cytotoxic T cells. Although epitopes are generally thought to be derived from non-self proteins, host-derived sequences that can be recognized are also classified as epitopes. Epitopes are at least 4 amino acids in length, preferably 4 to 30 amino acids, more preferably 5 to 20 amino acids, especially 5 to 15 amino acids. Epitopes may be linear or three-dimensional, typically consisting of amino acids that are distant from each other in the primary protein structure but become closely related in secondary and/or tertiary structure. Epitopes that are specifically recognized by B cells are called B cell epitopes.

The term "inverted terminal repeat" or "ITR" includes symmetrical nucleic acid sequences in the genome of adeno-associated viruses required for efficient replication. ITR sequences are found at each end of the AAV DNA genome. ITRs serve as the origins of replication for viral DNA synthesis and are essential cis components for generating integrating AAV vectors.

The term "light chain" includes an immunoglobulin light chain sequence from any organism, and unless otherwise specified includes the K and A light chains and VpreB, as well as surrogate light chains. Light chain variable domains typically include three light chain CDRs and four framework regions (FRs), unless otherwise specified. Generally, a full-length light chain includes, from the amino terminus to the carboxyl terminus, a variable domain including FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant region. A light chain variable domain is encoded by a light chain variable region gene sequence, generally comprising V<l> and J<l> segments, derived from a repertoire of V and J segments present in the germline. The sequences, locations, and nomenclature of V and J light chain segments for different organisms can be found in the IMGT database, www.imgt.org.

Light chains include those, for example, that do not selectively bind to a first or second epitope selectively bound by the epitope binding protein in which they appear. Light chains also include those that bind to and recognize, or assist the heavy chain or another light chain with binding and recognizing, one or more epitopes selectively bound by the epitope binding protein in which they appear. Common or universal light chains include those derived from a human V<k>1-39J<k>gene or a human V<k>3-20J<k>gene, and include somatically mutated (e.g., affinity matured) versions thereof. Illustrative human Vk1-39 gene segment, a human Vk3-20 gene segment, a human VA1-40 gene segment, a human VA1-44 gene segment, a human VA2-8 gene segment, a human VA2-14 gene segment, and a human VA3-21 gene segment, and include somatically mutated (e.g., affinity matured) versions thereof. Light chains can be produced that comprise a variable domain from one organism (e.g., human or rodent, e.g., rat or mouse; or bird, e.g., chicken) and a constant region from the same or a different organism (e.g., human or rodent, e.g., rat or mouse; or bird, e.g., chicken).

The term "about" or "approximately" includes being within a statistically significant range of a value. Such a range may be within an order of magnitude, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5% of a given value or range. The permissible variation encompassed by the term "about" or "approximately" depends on the particular system being studied, and can be readily appreciated by one skilled in the art.

The term "affinity tag" includes a polypeptide sequence that is a member of a specific binding pair, e.g., that specifically binds to another polypeptide sequence, e.g., an antibody paratope, with high affinity. Illustrative and non-limiting affinity tags include the hexahistidine tag, FLAG tag, Strep II tag, streptavidin-binding peptide (SBP) tag, calmodulin-binding peptide (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), S-tag, HA tag, and c-Myc tag. (Reviewed in Zhao et al. (2013) J. Analytical Meth. Chem. 1-8).

The term "capsid protein" includes a protein that forms part of the viral capsid. For adeno-associated viruses, the capsid proteins are usually referred to as VP1, VP2, and/or VP3, and each is encoded by a single cap gene. For AAV, the three AAV capsid proteins are produced in an overlapping manner from the decap open reading frame (ORF) by alternative mRNA splicing and/or alternative use of translation start codons, although all three proteins use a common stop codon. Warrington et al. (2004) J. Virol. 78:6595. AAV2 VP1 is typically translated from an ATG start codon (amino acid M1) on a 2.4 kb mRNA, whereas AAV2 VP2 and VP3 arise from a smaller 2.3 kb mRNA, using a weaker ACG start codon for production of VP2 (amino acid T138) and complete translation to the next available ATG codon (amino acid M203) for production of the more abundant capsid protein, VP3. Warrington, cited above; Rutledge et al. (1998) J. Virol. 72:309-19. The amino acid sequences of adeno-associated virus capsid proteins are well known in the art and are generally conserved, particularly over the dependoparvoviruses. See, Rutledge et al., cited above. For example, Rutledge et al. (1998), cited above, provide in Figure 4B amino acid sequence alignments for VP1, VP2, and VP3 capsid proteins of AAV2, AAV3, AAV4, and AAV6, where the start sites for each of the Vp1, VP2, and VP3 capsid proteins are indicated by arrows and the variable domains are boxed. Accordingly, although the amino acid positions provided herein may be provided in relation to the AAV VP1 capsid protein, and the amino acid positions provided herein that are not further specified refer to the AAV2 sequence of the major coat protein VP1 set forth as SEQ ID NO:1, one of skill in the art would be able to respectively and readily determine the position of that same amino acid within the AAV VP2 and/or VP3 capsid protein, and the corresponding position of the amino acids between different serotypes. Additionally, one of skill in the art could exchange domains between capsid proteins from different AAV serotypes for the formation of a "chimeric capsid protein."

Domain swapping between two AAV capsid protein constructs has been described for the generation of a "chimeric AAV capsid protein," see, e.g., Shenet al. (2007) Mol. Gene Therapy 15(11):1955-1962. A "chimeric AAV capsid protein" includes an AAV capsid protein that comprises amino acid sequences, e.g., domains, from two or more different AAV serotypes and that is capable of and/or forms an AAV-like viral capsid/viral particle. A chimeric AAV capsid protein is encoded by a chimeric AAV capsid gene, e.g., a nucleotide comprising a plurality, e.g., at least two, nucleic acid sequences, each of said plurality being identical to a portion of a capsid gene encoding a capsid protein from distinct AAV serotypes, and said plurality together encoding a functional chimeric AAV capsid protein. Reference to a chimeric capsid protein in relation to a specific AAV serotype indicates that the capsid protein comprises one or more domains from a capsid protein from that serotype and one or more domains from a capsid protein from a different serotype. For example, an AAV2 chimeric capsid protein includes a capsid protein comprising one or more domains from an AAV2 VP1, VP2, and/or VP3 capsid protein and one or more domains from a VP1, VP2, and/or VP3 capsid protein from a different AAV.

A "mosaic capsid" comprises at least two sets of VP1, VP2, and/or VP3 proteins, each set of which is encoded by a different cap gene.

In some embodiments, a mosaic capsid described herein comprises recombinant VP1, VP2, and/or VP3 proteins encoded by a genetically modified capgene with an insertion of a nucleic acid sequence encoding a heterologous epitope, and further comprises VP1, VP2, and/or VP3 proteins encoded by a reference capgene, e.g., a wild-type reference capgene encoding wild-type VP1, VP2, and/or VP3 proteins of the same AAV serotype as the recombinant VP1, VP2, and/or VP3 proteins, a control reference capgene encoding VP1, VP2, and/or VP3 proteins identical to the recombinant VP1, VP2, and VP3 proteins except for the absence of the heterologous epitope, a mutated wild-type reference capgene encoding substantially wild-type VP1, VP2, and/or VP3 proteins of the same AAV serotype that recombinant VP1, VP2, and/or VP3 proteins, except for a mutation (e.g., insertion, substitution, deletion), which mutation preferentially reduces the tropism of the wild-type VP1, VP2, and VP3 proteins. In some embodiments, the reference capsid protein is a chimeric reference protein comprising at least one domain from the VP1, VP2, and/or VP3 proteins of the same AAV serotype as the recombinant VP1, VP2, and/or VP3 proteins. In some embodiments, the reference capsid gene encodes a chimeric VP1, VP2, and/or VP3 protein.

The term "heavy chain" or "immunoglobulin heavy chain" includes an immunoglobulin heavy chain sequence, including the immunoglobulin heavy chain constant region sequence, from any organism. Heavy chain variable domains include three heavy chain CDRs and four FR regions, unless otherwise specified. Heavy chain fragments include CDRs, CDRs, and FRs, and combinations thereof. A typical heavy chain has, after the variable domain (from the N-terminus to the C-terminus), a C<h>1 domain, a hinge, a C<h>2 domain, and a C<h>3 domain. A functional fragment of a heavy chain includes a fragment that can specifically recognize an epitope (e.g., by recognizing the epitope with a Kd in the micromolar, nanomolar, or picomolar range), that is capable of being expressed and secreted from a cell, and that comprises at least one CDR. Heavy chain variable domains are encoded by a variable region nucleotide sequence, generally comprising Vh, Dh, and Jh segments derived from a repertoire of Vh, Dh, and Jh segments present in the germline. Sequences, locations, and nomenclature for heavy chain V, D, and J segments for different organisms can be found in the IMGT database, which is accessible via the Internet on the World Wide Web (www) at the URL "imgt.org".

The term "heavy chain-only antibody", "heavy chain-only antigen-binding protein", "single domain antigen-binding protein", "single domain binding protein" or the like refers to a monomeric or homodimeric immunoglobulin molecule comprising an immunoglobulin-like chain comprising a variable domain operably linked to a heavy chain constant region, which is incapable of associating with a light chain because the heavy chain constant region lacks a functional C<h>1 domain. Accordingly, the term "heavy chain only antibody", "heavy chain only antigen binding protein", "single domain antigen binding protein", "single domain binding protein" or the like encompasses both (i) a monomeric single domain antigen binding protein comprising one of the immunoglobulin-like chains comprising a variable domain operably linked to a heavy chain constant region lacking a functional C<h>1 domain, or (ii) a homodimeric single domain antigen binding protein comprising two immunoglobulin-like chains, each comprising a variable domain operably linked to a heavy chain constant region lacking a functional Ch1 domain. In various aspects, a homodimeric single domain antigen binding protein comprises two identical immunoglobulin-like chains, each comprising an identical variable domain operably linked to an identical heavy chain constant region lacking a functional C<h>1 domain. Additionally, each immunoglobulin-like chain of a single domain antigen binding protein comprises a variable domain, which may be derived from heavy chain variable region gene segments (e.g., V<h>, D<h>, J<h>), light chain gene segments (e.g., Vl, Jl), or a combination thereof, linked to a heavy chain constant region (Ch) gene sequence comprising a deletion or inactivating mutation in a Ch1 coding sequence (and, optionally, a hinge region) of a heavy chain constant region gene of, for example, IgG, IgA, IgE, IgD, or a combination thereof. A single domain antigen binding protein comprising a variable domain derived from heavy chain gene segments may be referred to as a "Vh single domain antibody" or a "Vh single domain antigen binding protein," see, for example, U.S. Pat. No. 8,754,287; U.S. Patent Publication Nos. 20140289876, 20150197553, 20150197554, 20150197555, 20150196015; 20150197556, and 20150197557. A single domain antigen binding protein comprising a variable domain derived from light chain gene segments may be referred to as a “Vl single domain antigen binding protein,” see, e.g., U.S. Publication No. 20150289489.

The term "light chain" includes an immunoglobulin light chain sequence from any organism, and unless otherwise specified includes kappa (k) and lambda (A) light chains and VpreB, as well as surrogate light chains. Light chain variable domains typically include three light chain CDRs and four framework regions (FRs), unless otherwise specified. Generally, a full-length light chain includes, from the amino terminus to the carboxyl terminus, a variable domain including FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant region amino acid sequence. Light chain variable domains are encoded by the nucleotide sequence of the light chain variable region, which typically comprises the V<l>light chain and J<l>light chain gene segments, derived from a repertoire of light chain V and J gene segments present in the germline. The sequences, locations and nomenclature of light chain V and J gene segments for different organisms can be found in the IMGT database, which can be accessed via the Internet on the World Wide Web (www) at the URL "imgt.org". Light chains include those, for example, that do not selectively bind to a first or second epitope selectively bound by the epitope binding protein on which they appear. Light chains also include those that bind and recognize, or assist the heavy chain with binding and recognizing, one or more epitopes selectively bound by the epitope binding protein on which they appear. Light chains also include those that bind and recognize, or assist the heavy chain with binding and recognizing, one or more epitopes selectively bound by the epitope binding protein on which they appear. Common or universal light chains include those derived from a human Vk1-39Jk5 gene or a human Vk3-20Jk1 gene, and include somatically mutated (e.g., affinity matured) versions thereof.

The term "operably linked," as used herein, includes a physical juxtaposition (e.g., in three-dimensional space) of components or elements that interact, directly or indirectly with one another, or coordinate with one another to participate in a biological event, which juxtaposition achieves or enables such interaction and/or coordination. To take but one example, a control sequence (e.g., an expression control sequence) in a nucleic acid is said to be "operably linked" to a coding sequence when it is located relative to the coding sequence such that its presence or absence affects the expression and/or activity of the coding sequence. In many embodiments, an "operable linkage" involves a covalent linkage of relevant components or elements to one another. Those skilled in the art will readily appreciate, however, that in some embodiments, a covalent linkage is not required to achieve an effective operable linkage. For example, in some embodiments, nucleic acid control sequences that are operably linked to coding sequences they control are contiguous with the nucleotide of interest. Alternatively or additionally, in some embodiments, one or more of such control sequences act in trans or at a distance to control a coding sequence of interest. In some embodiments, the term "expression control sequence" as used herein refers to polynucleotide sequences that are necessary and/or sufficient to effect expression and processing of coding sequences to which they are linked. In some embodiments, expression control sequences may be or comprise appropriate transcription initiation, termination, promoter, and/or enhancer sequences; effective RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance protein stability; and/or, in some embodiments, sequences that enhance protein secretion. In some embodiments, one or more control sequences are preferentially or exclusively active in a particular host cell or organism, or type thereof. To take just one example, in prokaryotes, control sequences typically include a promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, in many embodiments, control sequences typically include promoters, enhancers, and/or transcription termination sequences. Those skilled in the art will appreciate from the context that, in many embodiments, the term "control sequences" refers to components whose presence is essential for expression and processing, and in some embodiments, includes components whose presence is advantageous for expression (including, for example, leader sequences, targeting sequences, and/or fusion partner sequences).

The term "recombinant capsid protein" includes a capsid protein having at least one mutation compared to the corresponding capsid protein of the wild-type virus, which may be a reference and/or control virus for comparative study. A recombinant capsid protein includes a capsid protein comprising a heterologous epitope, which may be inserted into and/or presented by the capsid protein. "Heterologous" in this context means heterologous compared to the virus, from which the capsid protein is derived. The inserted amino acids may simply be inserted between two given amino acids of the capsid protein. An insertion of amino acids may also be accompanied by a deletion of certain amino acids of the capsid protein at the insertion site, for example, 1 or more amino acids of the capsid protein are replaced by 5 or more heterologous amino acids.

The terms "multispecific binding molecule" and "bispecific binding molecule", and the like generally and respectively refer to a binding molecule comprising at least two and only two non-identical binding partners, each binding partner specifically binding to a different epitope, either on two different molecules (e.g., different epitopes on two different immunogens) or on the same molecule (e.g., different epitopes on the same immunogen). Generally, one of the binding partners of a bispecific binding molecule herein specifically binds to a heterologous epitope presented by an AAV capsid protein and the second binding partner is specific for a protein, e.g., a cell surface marker, primarily and/or preferentially expressed by a target cell, e.g., a T cell marker (e.g., CD3, CD28, etc.). Bispecific binding molecules can be produced, for example, by combining binding partners that recognize different epitopes of the same immunogen. For example, nucleic acid sequences encoding binding components (e.g., light or heavy chain variable sequences) that recognize different epitopes can be fused to nucleic acid sequences encoding the same or different heavy chain constant regions, the same or different light chain constant regions, or respectively a heavy chain constant region and a light chain constant region, and such sequences can be expressed in a cell as a multispecific antigen binding protein in a format that is similar to a Fab structure, scFab structure, a diabody structure, a scFv structure, a scFv-Fc structure, a scFv zipper structure, a typical antibody-like tetrameric structure including the cognate universal light chain, a tetrameric structure comprising a typical bivalent antibody including the cognate universal light chain and/or an additional binding component (e.g., scFv, scFv-zipper, scFab, etc.) attached to one or both of the heavy chains (e.g., at the N-terminus and/or at the N-terminus). C) and/or to one or both light chains (e.g. at the N- and/or C-terminus). The different formats of multispecific, particularly bispecific, binding molecules are well known, see, for example, Brinkmann and Konterman (2017)mAbs9:182–212.

An exemplary multispecific binding molecule has two heavy chains, each of which has heavy chain CDRs, followed by a Ch1 domain (N-terminus to C-terminus), a hinge, a Ch2 domain, and a Ch3 domain, and an immunoglobulin light chain that does not confer epitope binding specificity but that can associate with each heavy chain (e.g., a common light chain), or that can associate with each heavy chain and that can bind to one or more epitopes, via the epitope binding regions of the heavy chain, or that can associate with each heavy chain and allow binding of one or both of the heavy chains to one or both epitopes. In some embodiments, a multispecific binding molecule comprises: (1) an immunoglobulin heavy chain variable domain operably linked to a first heavy chain constant region comprising a first CH3 amino acid sequence of a human IgG selected from IgG1, IgG2, IgG4, and a combination thereof; and (2) an immunoglobulin heavy chain variable domain, wherein the second immunoglobulin heavy chain variable domain is operably linked to a second heavy chain constant region comprising a second CH3 amino acid sequence of human IgG selected from IgG1, IgG2, IgG4, and a combination thereof, wherein the first or second heavy chain variable domain (with or without a cognate light chain) binds to a heterologous epitope as described herein and the other heavy chain variable domain (with or without a cognate light chain) binds to a receptor on a target cell, and wherein the first heavy chain constant region is associated with the second constant chain region in a manner that provides for easier isolation of the multispecific binding protein, e.g., wherein the first and second heavy chain constant regions form a "button-in-buttonhole" (KIH) format or wherein the second CH3 amino acid sequence comprises a modification that reduces or eliminates binding of the second CH3 amino acid sequence to Protein A (see, e.g., U.S. Patent No. 8,586,713). In some embodiments, a multispecific binding molecule comprises: (1) an immunoglobulin heavy chain variable domain operably linked to a first heavy chain constant region comprising a first CH3 amino acid sequence of a human IgG selected from IgG1, IgG2, IgG4, and a combination thereof; and (2) an immunoglobulin heavy chain variable domain, wherein the second immunoglobulin heavy chain variable domain is operably linked to a second heavy chain constant region comprising a second CH3 amino acid sequence of human IgG selected from IgG1, IgG2, IgG4, and a combination thereof, wherein the first and second heavy chain variable domains (with or without a cognate light chain) bind the same or different antigens, wherein the first or second heavy chain constant region is modified to further comprise an additional binding domain (e.g., a scFv or Fv that binds a heterologous epitope as described herein, e.g., wherein the additional binding domain is linked to the C- or N-terminus of one or both heavy chains), and wherein the first heavy chain constant region is associated with the second constant chain region in a manner that provides for easier isolation of the multispecific binding protein, e.g., wherein the first and second heavy chain constant regions form a scaffold format. "button-in-button" (KIH) or wherein the second CH3 amino acid sequence comprises a modification that reduces or eliminates binding of the second CH3 amino acid sequence to Protein A. In some embodiments wherein the second CH3 amino acid sequence comprises reduced or eliminated binding to Protein A, the second CH3 amino acid sequence comprises an H95R modification (according to IMGT exon numbering; H435R according to EU numbering). In one embodiment, the second CH3 amino acid sequence further comprises a Y96F modification (according to IMGT exon numbering; H436F according to EU numbering). In another embodiment, the second CH3 amino acid sequence comprises both an H95R modification (according to IMGT exon numbering; H435R according to EU numbering) and a Y96F modification (according to IMGT exon numbering; H436F according to EU numbering). In some embodiments, the second amino acid sequence of CH3 is from a modified human IgG1 and further comprises a mutation selected from the group consisting of D16E, L18M, N44S, K52N, V57M, and V82I (IMGT; D356E, L38M, N384S, K392N, V397M, and V422I according to EU). In some embodiments, the second amino acid sequence of CH3 is from a modified human IgG2 and further comprises a mutation selected from the group consisting of N44S, K52N, and V82I (IMGT: N384S, K392<n>and V422I according to EU). In some embodiments, the second CH3 amino acid sequence is from a modified human IgG4 and further comprises a mutation selected from the group consisting of Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (IMGT: Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I according to EU). In some embodiments, the heavy chain constant region amino acid sequence is a non-human constant region amino acid sequence, and the heavy chain constant region amino acid sequence comprises one or more of any of the types of modifications described above.

In different embodiments, Fc domains are modified to alter Fc receptor binding, which in turn affects effector function. In some embodiments, an engineered heavy chain constant (CH) region, which includes the Fc domain, is chimeric. As such, a chimeric CH region combines CH domains derived from more than one immunoglobulin isotype. For example, a chimeric CH region comprises part or all of a CH2 domain derived from a human IgG1, human IgG2, or human IgG4 molecule, in combination with part or all of a CH3 domain derived from a human IgG1, human IgG2, or human IgG4 molecule. In some embodiments, a chimeric CH region contains a chimeric hinge region. For example, a chimeric hinge can comprise an "upper hinge" amino acid sequence (amino acid residues at positions 216 to 227 according to EU numbering; amino acid residues at positions 226 to 240 according to Kabat numbering) derived from a hinge region of a human IgG1, a human IgG2, or a human IgG4, in combination with a "lower hinge" sequence (amino acid positions 228 to 236 according to EU numbering; amino acid positions 241 to 249 according to Kabat numbering) derived from a hinge region of a human IgG1, a human IgG2, or a human IgG4. In some embodiments, the chimeric hinge region comprises amino acid residues from an upper hinge of human IgG1 or human IgG4 and amino acid residues from a lower hinge of human IgG2.

In some embodiments, the Fc domain can be engineered to activate all, some, or none of the normal Fc effector functions, without affecting the desired pharmacokinetic properties of the Fc-containing protein (e.g., antibody). For examples of proteins comprising chimeric CH regions and having altered effector functions, see WO2014022540.

The term "target cells" includes any cell in which expression of a nucleotide of interest is desired. Preferably, the target cells display a protein, e.g., a receptor, on their surface that allows the cell to be targeted with a retargeting ligand, as described below. Preferably, the target protein, e.g., the receptor is specific for the target cell, e.g., is a "cell-specific marker," "cell-specific antigen," or the like. The term "cell-specific marker," "cell-specific antigen," "organ-specific marker," "tissue-specific marker," or the like refers to and includes those proteins whose expression is enriched by the cell, tissue, and/or organ for which it is a specific marker. "Enriched" in the context of protein expression refers to and includes expression or overexpression of the cell/tissue/organ-specific protein primarily, preferentially, or solely, by the cell/tissue/organ for which the protein is a specific marker, although such marker may also be expressed by other cells/tissues/or organs at trace levels. The Human Protein Atlas can be used to determine whether a protein is a cell/tissue/organ-specific marker and also provides a repository of cell/tissue/organ-specific proteins. See, www.proteinatlas.org; see also Uhlenet al. (2010) Nat. Biotech. 28:1248-50.

The term "transduction" or "infection" or the like refers to the introduction of a nucleic acid into a target cell by a viral vector. The term efficiency in relation to transduction or the like, e.g., "transduction efficiency" refers to the fraction (e.g., percentage) of cells expressing a nucleotide of interest after incubation with a given number of viral vectors comprising the nucleotide of interest. Well-known methods for determining transduction efficiency include fluorescence-activated cell sorting of cells transduced with a fluorescent reporter gene, PCR for expression of the nucleotide of interest, etc.

The term "wild type" as used herein includes an entity that has a structure and/or activity as found in nature in a "normal" (as opposed to mutant, pathological, altered, etc.) state or context. Those skilled in the art will appreciate that wild type viral vectors, e.g. wild type capsid proteins, can be used as a reference viral vector in comparative studies. Generally, a reference viral capsid protein/capsid/vector is identical to the test viral capsid protein/capsid/vector except for the change for which the effect is to be tested. For example, to determine the effect, e.g., on transduction efficiency, of inserting a heterologous epitope into a test viral vector, the transduction efficiencies of the test viral vector (in the absence or presence of an appropriate multispecific binding molecule) can be compared to the transduction efficiencies of a reference viral vector (in the absence or presence of an appropriate multispecific binding molecule if necessary) that is identical to the test viral vector in all respects (e.g., additional mutations, nucleotide of interest, number of viral vectors and target cells, etc.) except for the presence of a heterologous epitope.

Recombinant virus capsid proteins and AAV nucleic acids and vectors

In some embodiments, a recombinant AAV capsid protein as described herein is an Ad capsid protein, e.g., a capsid protein from an Ad serotype selected from the group consisting of Ad1, Ad2, Ad3, Ad4, Ad5, Ad6, and Ad7. In some embodiments, a recombinant AAV capsid protein is derived from an Ad2 capsid gene. In some embodiments, a recombinant AAV capsid protein is derived from an Ad5 capsid gene. In some embodiments, a recombinant Ad AAV capsid protein as described herein comprises a heterologous epitope in a fiber protein domain, e.g., at the carboxyl terminus of the fiber protein, fiber knob, and/or HI loop of the fiber knob.

In some embodiments, a recombinant AAV capsid protein derived from an adeno-associated virus (AAV) capsid gene is a genetically modified capsid protein of an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. In some embodiments, the recombinant AAV capsid protein is derived from an AAV2 capsid gene, an AAV6 capsid gene, an AAV8 capsid gene, or an AAV9 capsid gene. In some embodiments, the recombinant AAV capsid protein is derived from an AAV2 capsid gene, for example, it is a genetically modified AAV2 VP1 capsid protein, the amino acid sequence of which for the wild type is respectively set forth as SEQ ID NO:1. In some embodiments, the recombinant AAV capsid protein is derived from an AAV8 capsid gene, for example, it is a genetically modified AAV8 VP1 capsid protein, the amino acid sequence of which for the wild type is respectively set forth as SEQ ID NO:21. In some embodiments, the recombinant AAV capsid protein is derived from an AAV9 capsid gene, for example, it is a genetically modified AAV9 VP1 capsid protein, the amino acid sequence of which for the wild type is respectively set forth as SEQ ID NO:5. In some embodiments, the recombinant AAV capsid protein is derived from an AAV6 capsid gene, e.g., is a genetically modified AAV6 VP1 capsid protein. In some embodiments, a heterologous epitope is inserted into I-453 of an AAV9 capsid protein.

Generally, a recombinant AAV capsid protein as described herein comprises a heterologous epitope inserted into and/or presented by the AAV capsid protein such that the heterologous epitope reduces and/or abrogates the natural tropism of the AAV capsid protein or the capsid comprising it. In some embodiments, the heterologous epitope is inserted into a region of the AAV capsid protein involved with the natural tropism of the wild-type reference capsid protein, e.g., a region of the capsid protein involved with a cellular receptor. In some embodiments, the heterologous epitope is inserted into and/or presented by a knob domain of an Ad fiber protein. In some embodiments, the heterologous epitope is inserted into and/or presented by the HI loop of an Ad fiber protein. In some embodiments, the heterologous epitope is inserted after an amino acid position selected from the group consisting of G-453 of the AAV2 capsid VP1 protein, N-587 of the AAV2 capsid VP1 protein, Q-585 of the AAV6 capsid VP1 protein, G-453 of the AAV9 capsid VP1 protein, and A-589 of the AAV9 capsid VP1 protein. In some embodiments, the heterologous epitope is inserted and/or presented between amino acids N-587 and R-588 of an AAV2 VP1 capsid. In some embodiments, a recombinant AAV capsid, AAV vector comprising a recombinant AAV capsid, and/or compositions comprising a recombinant AAV capsid comprise an amino acid sequence set forth as SEQ ID NO:2. In some embodiments, a recombinant AAV capsid, AAV vector comprising a recombinant AAV capsid, and/or compositions comprising a recombinant AAV capsid comprise an amino acid sequence set forth as SEQ ID NO:4. In some embodiments, a recombinant AAV capsid, AAV vector comprising a recombinant AAV capsid, and/or compositions comprising a recombinant AAV capsid comprise an amino acid sequence encoded by the nucleic acid sequence set forth as SEQ ID NO:25. In some embodiments, a recombinant AAV capsid, AAV vector comprising a recombinant AAV capsid, and/or compositions comprising a recombinant AAV capsid comprise an amino acid sequence encoded by the nucleic acid sequence set forth as SEQ ID NO:26. In some embodiments, a recombinant AAV capsid, AAV vector comprising a recombinant AAV capsid, and/or compositions comprising a recombinant AAV capsid comprise an amino acid sequence encoded by the nucleic acid sequence set forth as SEQ ID NO:27. Additional suitable insertion sites identified using AAV2 are well known in the art (Wue et al. (2000) J. Virol. 74:8635-8647) and include I-1, I-34, I-138, I-139, I-161, I-261, I-266, I-381, I-447, I-448, I-459, I-471, I-520, I-534, I-570, I-573, I-584, I-587, I-588, I-591, I-657, I-664, I-713, and I-716. A recombinant virus capsid protein as described herein may be an AAV2 capsid protein comprising a heterologous epitope inserted at a position selected from the group consisting of I-1, I-34, I-138, I-139, I-161, I-261, I-266, I-381, I-447, I-448, I-459, I-471, I-520, I-534, I-570, I-573, I-584, I-587, I-588, I-591, I-657, I-664, I-713, I-716, and a combination thereof. Additional suitable insertion sites identified through the use of additional AAV serotypes are well known and include I-587 (AAV1), I-589 (AAV1), I-585 (AAV3), I-585 (AAV4), and I-585 (AAV5). In some embodiments, a recombinant AAV capsid protein as described herein can be an AAV2 capsid protein comprising a heterologous epitope inserted at a position selected from the group consisting of I-587 (AAV1), I-589 (AAV1), I-585 (AAV3), I-585 (AAV4), I-585(AAV5), and a combination thereof.

The nomenclature used I-n°n°n° herein refers to the insertion site with n°n°n° naming the number of amino acids relative to the VP1 protein of an AAV capsid protein, however, such insertion may be located directly at the N- or C-terminus, preferably the C-terminus of an amino acid in the sequence of 5 N- or C-terminal amino acids of the given amino acid, preferably 3, more preferably 2, especially 1 N- or C-terminal amino acid of the given amino acid. Additionally, the positions referenced herein are relative to the VP1 protein encoded by an AAV capsid gene, and the corresponding positions (and their mutations) can be readily identified for the VP2 and VP3 capsid proteins encoded by the capsid gene by performing a sequence alignment of the VP1, VP2, and VP3 proteins encoded by the reference AAV capsid gene.

Accordingly, an insertion at the corresponding position of the nucleic acid encoding one of these sites of the capsid gene leads to an insertion into VP1, VP2 and/or VP3, since the capsid proteins are encoded by overlapping reading frames of the same gene with staggered start codons. Thus, for AAV2, for example, according to this nomenclature, insertions between amino acids 1 and 138 are only inserted into VP1, insertions between 138 and 203 are inserted into VP1 and VP2, and insertions between 203 and the C-terminus are inserted into VP1, VP2 and VP3, which is of course also the case for insertion site I-587. The present invention therefore encompasses AAV structural genes with corresponding insertions into the VP1, VP2 and/or VP3 proteins.

Additionally, due to the high conservation of at least large stretches and the large number of closely related family members, corresponding insertion sites for AAVs other than the listed AAV can be identified by performing an amino acid alignment or by comparing capsid structures. See, e.g., Rutledge et al. (1998) J. Virol. 72:309-19 and U.S. Patent No. 9,624,274 for illustrative alignments of different AAV capsid proteins.

In some compositions disclosed herein consisting of recombinant AAV capsid (e.g., in the absence of a multispecific binding molecule), the recombinant AAV capsid protein is an AAV2 capsid VP1 protein with a heterologous epitope inserted at a 1587 site, wherein the heterologous epitope does not comprise an Arg-Gly-Asp (RGD) motif, an NGR motif, or c-myc. In some compositions disclosed herein consisting of recombinant AAV capsid (e.g., in the absence of a multispecific binding molecule), the recombinant AAV capsid protein is a VP1 capsid protein with a heterologous epitope inserted between T-448 and N-449, wherein the heterologous epitope does not comprise c-myc. In some compositions disclosed herein consisting of recombinant AAV capsid (e.g., in the absence of a multispecific binding molecule), the recombinant AAV capsid protein is a VP1 capsid protein with a heterologous epitope inserted at an I-447 site, wherein the heterologous epitope does not comprise L14 or HA.

In some compositions comprising recombinant AAV capsid (e.g., further comprising a multispecific binding molecule), the recombinant AAV capsid protein is a VP1 capsid protein with a heterologous epitope inserted at an I-587 site, wherein the heterologous epitope comprises an Arg-Gly-Asp (RGD) motif, an NGR motif, or c-myc. In some compositions disclosed herein comprising recombinant AAV capsid (e.g., further comprising a multispecific binding molecule), the AAV capsid is a VP1 capsid, the heterologous epitope comprises c-myc, and the heterologous epitope is inserted between T-448 and N-449, or between N-587 and R-588. In some compositions disclosed herein comprising recombinant AAV capsid (e.g., further comprising a multispecific binding molecule), the recombinant AAV capsid protein is a VP1 capsid protein with a heterologous epitope inserted at an I-447 site, wherein the heterologous epitope comprises L14 or HA. In some compositions disclosed herein comprising recombinant AAV capsid (e.g., in the presence of a multispecific binding molecule), the recombinant AAV capsid protein is a VP1 capsid protein with a heterologous epitope inserted between T-448 and N-449, wherein the heterologous epitope comprises c-myc. US Patent No. 9,624,274 describes I-453 of an AAV capsid protein as a suitable insertion site for a heterologous epitope.

In some embodiments, insertion (presentation) of the heterologous epitope abrogates the natural tropism of the AAV vector, e.g., transduction of a cell naturally permissive to infection by wild-type reference AAV vectors and/or a target cell is undetectable in the absence of an appropriate multispecific binding molecule. In some embodiments, insertion (presentation) of the heterologous epitope reduces the natural tropism of the AAV vector, e.g., as compared to transduction of a cell naturally permissive to infection by wild-type reference AAV vectors. In some embodiments, insertion (presentation) of the heterologous epitope reduces the natural tropism of the AAV vector by at least 5%. In some embodiments, insertion (presentation) of the heterologous epitope reduces the natural tropism of the AAV vector by at least 5%. In some embodiments, the insertion (presentation) of the heterologous epitope reduces the natural tropism of the AAV vector by at least 10%. In some embodiments, the insertion (presentation) of the heterologous epitope reduces the natural tropism of the AAV vector by at least 20%. In some embodiments, the insertion (presentation) of the heterologous epitope reduces the natural tropism of the AAV vector by at least 30%. In some embodiments, the insertion (presentation) of the heterologous epitope reduces the natural tropism of the AAV vector by at least 40%. In some embodiments, the insertion (presentation) of the heterologous epitope reduces the natural tropism of the AAV vector by at least 50%. In some embodiments, the insertion (presentation) of the heterologous epitope reduces the natural tropism of the AAV vector by at least 60%. In some embodiments, the insertion (presentation) of the heterologous epitope reduces the natural tropism of the AAV vector by at least 70%. In some embodiments, the insertion (presentation) of the heterologous epitope reduces the natural tropism of the AAV vector by at least 80%. In some embodiments, the insertion (presentation) of the heterologous epitope reduces the natural tropism of the AAV vector by at least 90%. In some embodiments, the insertion (presentation) of the heterologous epitope reduces the natural tropism of the AAV vector by at least 95%. In some embodiments, the insertion (presentation) of the heterologous epitope reduces the natural tropism of the AAV vector by at least 90%. In these embodiments, where the insertion (presentation) of the heterologous epitope does not abrogate the natural tropism of the recombinant AAV capsids, the natural tropism of said recombinant AAV capsids may be abrogated by a second, different mutation. For example, in one embodiment, a recombinant AAV capsid protein as described herein may be derived from an AAV9 capsid gene, comprise a heterologous epitope, and may further comprise a mutation, for example, a W503A mutation.

This separation of the virus from its natural host cell is important, especially if systemic versus local or locoregional delivery of AAV vectors is intended, since uptake of AAV vectors by natural host cells limits the effective dose of AAV vectors. In the case of AAV2 and AAV6, HSPG is reported to be the major receptor for viral uptake in a large number of cells, especially liver cells. For AAV2, HSPG binding activity is dependent on a cluster of 5 basic amino acids, R484, R487, R585, R588, and K532 (Kernet al.,(2003)J Virol.77(20):11072-81). Recently it was reported that substitution of the amino acid K531E from lysine to glutamate leads to abolition of the ability of AAV6 to bind heparin or HSPG ((Wuet al.,2006)J. of Virology80(22):11393-11397). Consequently, preferred point mutations are those that reduce the transduction activity of the AAV vector for a given target cell mediated by the natural receptor by at least 50%, preferably at least 80%, especially at least 95%, in the case of HSPG as the primary receptor, the binding of AAV vectors to HSPG.

Accordingly, other preferred mutations for HSPG-binding AAV vectors are those mutations that decrease or replace a basic amino acid such as R, K or H, preferably R or K that is involved in HSPG binding of the respective virus, by a non-basic amino acid, such as A, D, G, Q, S and T, preferably A or an amino acid that is present at the corresponding position of a different but highly conserved AAV serotype that lacks said basic amino acid at this position. Accordingly, preferred amino acid substitutions are R-484-A, R-487-A, R-487-G, K-532-A, K-532-D, R-585-A, R-585-S, R-585-Q, R-585-A or R-588-T, especially R-585-A and/or R-588-A for AAV2, and K-531-A or K-531-E for AAV6. An especially preferred embodiment of the invention are AAV2 capsid protein mutants additionally containing the two point mutations R-585-A and R-588-A, since these two point mutations are sufficient to abrogate HSPG binding activity to a large extent. These point mutations allow efficient separation of HSPG-expressing cells which, for targeting purposes, increases the specificity of the respective mutant virus for its new target cell.

One embodiment of the present invention is a multimeric structure comprising a recombinant AAV capsid protein of the present invention. A multimeric structure comprises at least 5, preferably at least 10, more preferably at least 30, most preferably at least 60 recombinant AAV capsid proteins comprising a heterologous epitope as described herein. They may form regular AAV capsids (empty AAV particles) or AAV vectors (capsids encapsulating a nucleotide of interest). The formation of AAV vectors capable of packaging a viral genome is a highly preferred feature for the use of the recombinant AAV capsids described herein as AAV vectors.

One embodiment of the present invention is a nucleic acid encoding a capsid protein as described above. The nucleic acid is preferably a vector comprising the claimed nucleic acid sequence. Nucleic acids, especially vectors, are necessary to recombinantly express the capsid proteins of this invention.

A further embodiment of the present invention is the use of at least one recombinant AAV capsid protein and/or a nucleic acid encoding the same, preferably at least one multimeric structure (e.g., AAV vector) for manufacture and use as a gene transfer vector.

Heterologous epitopes

Generally, a recombinant AAV protein and/or an AAV vector comprising the recombinant AAV capsid comprises a heterologous epitope, which allows for retargeting of the AAV vector, e.g., via a multispecific binding molecule. In some embodiments, a heterologous epitope is a B cell epitope, e.g., that is between about 1 amino acid and about 35 amino acids in length, and forms a binding pair with an antibody paratope, e.g., an immunoglobulin variable domain. In some embodiments, the heterologous epitope comprises an affinity tag.

A large number of tags are known in the art. (See, for example: Nilsson et al. (1997) "Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins" Protein Expression and Purification 11: 1-16, Terpe et al. (2003) "Overview of tag protein fusions: From molecular and biochemical fundamentals to commercial systems" Applied Microbiology and Biotechnology 60:523-533, and references therein). Affinity tags include, but are not limited to, a polyhistidine tag (e.g., a His-6, His-8, or His-10 tag) that binds to immobilized divalent cations (e.g., Ni 2+ ), a biotin moiety (e.g., in a biotinylated polypeptide sequence in vivo) that binds to immobilized avidin, a GST (glutathione S-transferase) sequence that binds to immobilized glutathione, an S-tag that binds to immobilized protein S, an antigen that binds to an immobilized antibody or domain or fragment thereof (including, for example, T7, myc, FLAG, and B tags that bind to corresponding antibodies), a FLASH tag (a high-affinity tag that couples to specific arsenic-based moieties), a receptor or receptor domain that binds to an immobilized ligand (or vice versa), protein A or a derivative thereof (e.g., Z) that binds to IgG immobilized, maltose binding protein (MBP) that binds immobilized amylose, an albumin binding protein that binds immobilized albumin, a chitin binding domain that binds immobilized chitin, a calmodulin binding peptide that binds immobilized calmodulin, and a cellulose binding domain that binds immobilized cellulose. Another exemplary tag is a SNAP tag, marketed by Covalys (www.covalvs.com). In some embodiments, a heterologous epitope disclosed herein comprises an affinity tag recognized solely by one antibody paratope. In some embodiments, a heterologous epitope disclosed herein comprises an affinity tag recognized by an antibody paratope and other specific binding pairs.

In some aspects, the heterologous epitope and/or affinity tag does not form a binding pair with an immunoglobulin constant domain. In some aspects, the heterologous epitope and/or affinity tag does not form a binding pair with a metal ion, e.g., Ni2+, Co2+, Cu2+, Zn2+, Fe3+, etc. In some embodiments, the heterologous epitope is not a polypeptide selected from the group consisting of streptavidin, Strep II, HA, L14, 4C-RGD, L<h>and Protein A.

In some embodiments, the affinity tag is selected from the group consisting of FLAG (SEQ ID NO:7), HA (SEQ ID NO:8), and c-myc (EQKL<i>S<e>EDL; SEQ ID NO: 6). In some embodiments, the heterologous epitope is c-myc.

In some embodiments, a recombinant AAV capsid as described herein comprises the amino acid sequence EQKLISEEDL (set forth as SEQ ID NO:6) flanked by and/or operably linked to at least 5 contiguous amino acids of an AAV VP1 capsid protein. In some embodiments, a recombinant AAV capsid as described herein comprises the amino acid sequence EQKLISEEDL (set forth as SEQ ID NO:6) flanked by and/or operably linked to at least 5 contiguous amino acids of an AAV2 VP1 capsid protein. In some embodiments, a recombinant AAV capsid as described herein comprises EQKLISEEDL (set forth as SEQ ID NO:6) inserted between N587 and R588 of an AAV2 VP1 capsid protein. In some embodiments, a recombinant AAV capsid protein as described herein comprises an amino acid sequence set forth as SEQ ID NO:2. In some embodiments, a recombinant AAV capsid protein as described herein comprises an amino acid sequence set forth as SEQ ID NO:4. In some embodiments, a recombinant AAV capsid, AAV vector comprising a recombinant AAV capsid, and/or compositions comprising a recombinant AAV capsid comprise an amino acid sequence encoded by the nucleic acid sequence set forth as SEQ ID NO:25. In some embodiments, a recombinant AAV capsid, AAV vector comprising a recombinant AAV capsid, and/or compositions comprising a recombinant AAV capsid comprise an amino acid sequence encoded by the nucleic acid sequence set forth as SEQ ID NO:26. In some embodiments, a recombinant AAV capsid, the AAV vector comprising a recombinant AAV capsid, and/or the compositions comprising a recombinant AAV capsid comprise an amino acid sequence encoded by the nucleic acid sequence set forth as SEQ ID NO:27.

In some embodiments, the heterologous epitope comprises an affinity tag and one or more linkers. In some embodiments, the heterologous epitope comprises an affinity tag flanked by a linker, e.g., the heterologous epitope comprises from N-terminus to C-terminus a first linker, an affinity tag, and a second linker. In some embodiments, the first and second linkers are each independently at least one amino acid in length. In some embodiments, the first and second linkers are identical.

Generally, a heterologous epitope as described herein, e.g., an affinity tag alone or together with one or more linkers, is between about 5 amino acids and 35 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is at least 5 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is 6 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is 7 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is 8 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is 9 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is 10 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is 11 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is 12 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is 13 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is 14 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is 15 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is 16 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is 17 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is 18 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is 19 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is 20 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is 21 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is 22 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is 23 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is 24 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is 25 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is 26 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is 27 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is 28 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is 29 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is 30 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is 31 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is 32 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is 33 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is 34 amino acids in length. In some embodiments, the heterologous epitope (alone or together with one or more linkers) is 35 amino acids in length.

Redirect leftovers

An AAV vector as described herein has reduced to abolished transduction capabilities in the absence of a multispecific binding molecule, specifically a multispecific binding molecule comprising (i) an antibody paratope that specifically binds to the epitope and (ii) a retargeting ligand that specifically binds to a receptor, which may be conjugated to the surface of a bead (e.g., for purification) or expressed by a target cell. Accordingly, a multispecific binding molecule comprising (i) an antibody paratope that specifically binds to the epitope and (ii) a retargeting ligand that specifically binds to a receptor redirects the AAV vector. Such "retargeting" may include a scenario in which the wild-type AAV vector is targeted to multiple cells within a tissue and/or to multiple organs within an organism, whose broad targeting to the tissue or organs is reduced or abrogated by insertion of the heterologous epitope, and whose retargeting to more specific cells in the more specific tissue or organ in the organism is achieved with the multispecific binding molecule. Such retargeting may also include a scenario in which the wild-type AAV vector is targeted to a tissue, whose targeting to the tissue is reduced or abrogated by insertion of the heterologous epitope, and whose retargeting to a completely different tissue is achieved with the multispecific binding molecule. An antibody paratope as described herein generally comprises at least one complementarity determining region (CDR) that specifically recognizes the heterologous epitope, e.g., a CDR3 region of a heavy and/or light chain variable domain. In some embodiments, a multispecific binding molecule comprises an antibody (or portion thereof) comprising the antibody paratope that specifically binds to the heterologous epitope. For example, a multispecific binding molecule may comprise a single domain heavy chain variable region or a single domain light chain variable region, wherein the single domain heavy chain variable region or the single domain light chain variable region comprises an antibody paratope that specifically binds to the heterologous epitope. In some embodiments, a multispecific binding molecule may comprise an Fv region, for example, a multispecific binding molecule may comprise an scFv, which comprises an antibody paratope that specifically binds to the heterologous epitope. In some embodiments, a multispecific binding molecule as described herein comprises an antibody paratope that specifically binds to c-myc.

In some embodiments, a multispecific binding molecule as described herein comprises an antibody paratope that specifically binds to c-myc, said paratope comprising the scFv, the heavy and light chain variable domains of the scFv, and/or the amino acid sequence set HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3, encoded by the nucleic acid sequence set forth as SEQ ID NO:28, e.g., the scFv comprises the amino acid sequence set forth as SEQ ID NO: 37. In some embodiments, a multispecific binding molecule as described herein comprises an Fv or scFv encoded by the nucleic acid sequence set forth as SEQ ID NO:28.

Accordingly, the present invention includes antibodies, antigen-binding antibody fragments, and multispecific binding proteins that specifically bind to c-myc, wherein the antibodies, antibody fragments, and multispecific binding proteins comprise a paratope comprising a heavy chain variable region (HCVR) comprising SEQ ID NO:29 and a light chain variable region (LCVR) comprising SEQ ID NO:30. The present invention also includes antibodies, antigen-binding antibody fragments, and/or multispecific binding proteins comprising a paratope that specifically binds c-Myc, wherein the paratope comprises a heavy chain complementarity determining region 1 (HCDR1) comprising SEQ ID NO:31, a HCDR2 comprising SEQ D NO:32, a HCDR3 comprising SEQ ID NO:33, a light chain complementarity determining region 1 (LCDR1) comprising SEQ ID NO:34, a LCDR2 comprising SEQ ID NO:35, and a LCDR3 comprising SEQ ID NO:36.

The present invention may employ antibodies, antigen-binding fragments thereof, and/or multispecific binding proteins comprising a paratope comprising an HCVR comprising an amino acid sequence set forth as SEQ ID NO:29, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

The present invention may employ antibodies, antigen-binding fragments thereof, and/or multispecific binding proteins comprising a paratope comprising an LCVR comprising an amino acid sequence set forth as SEQ ID NO:30, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

The present invention may employ antibodies, antigen-binding fragments thereof, and/or multispecific binding proteins comprising a paratope comprising an amino acid sequence pair of HCVR and LCVR (HCVR/LCVR) comprising the HCVR amino acid sequence set forth as SEQ ID NO:29 paired with the LCVR amino acid sequence set forth as SEQ ID NO:30. In some embodiments, the HCVR/LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NO: 29/30.

The present invention may employ antibodies, antigen-binding fragments thereof, and/or multispecific binding proteins comprising a paratope comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence set forth as SEQ ID NO:31 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention may employ antibodies, antigen-binding fragments thereof, and/or multispecific binding proteins comprising a paratope comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence set forth as SEQ ID NO:32, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention may employ antibodies, antigen-binding fragments thereof, and/or multispecific binding proteins comprising a paratope comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence set forth as SEQ ID NO:33, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention may employ antibodies, antigen-binding fragments thereof, and/or multispecific binding proteins comprising a paratope comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence set forth as SEQ ID NO:34, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention may employ antibodies, antigen-binding fragments thereof, and/or multispecific binding proteins comprising a paratope comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence set forth as SEQ ID NO:35, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention may employ antibodies, antigen-binding fragments thereof, and/or multispecific binding proteins comprising a paratope comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence set forth as SEQ ID NO:36, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention may employ antibodies, antigen-binding fragments thereof, and/or multispecific binding proteins comprising a paratope comprising an amino acid sequence pair of HCDR3 and LCDR3 (HCDR3/LCDR3) comprising the HCDR3 amino acid sequence set forth as SEQ ID NO:33 paired with the LCDR3 amino acid sequence set forth as SEQ ID NO:36. In some embodiments, the HCDR3/LCDR3 amino acid sequence pair is set forth as SEQ ID NO: 33/36.

The present invention may employ antibodies, antigen-binding fragments thereof, and/or multispecific binding proteins comprising a paratope comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) encoded by the nucleotide sequence set forth as SEQ ID NO:28. In certain embodiments, the set of amino acid sequences HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 is set forth as SEQ ID NO: 31-32-33-34-35-36.

In a related embodiment, the present invention may employ antibodies, antigen-binding fragments thereof, and/or multispecific binding proteins comprising a paratope comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR/LCVR amino acid sequence pair set forth as SEQ ID NO: 29/30. Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and may be used to identify CDRs within the specific HCVR and/or LCVR amino acid sequences disclosed herein. Illustrative conventions that may be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the mAb definition. Broadly speaking, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the mAb definition is a compromise between the Kabat and Chothia approaches. See, for example, Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikanie et al., J. Mol. Biol. 273:927–948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268–9272 (1989). Public databases are also available for identifying CDR sequences within an antibody.

The present invention may employ nucleic acid molecules encoding anti-myc antibodies or portions thereof.

A multispecific binding molecule as described herein further comprises a retargeting ligand, in addition to a paratope (e.g., an antibody or portion thereof) that specifically binds to the heterologous epitope inserted/presented by a recombinant AAV capsid protein. In some embodiments, the retargeting ligand binds to a protein expressed on the surface of a cell, e.g., a cell surface protein on a eukaryotic (human) cell, e.g., a target cell. There are a large number of suitable cell surface proteins, e.g., cell surface receptors, that can be targeted by a retargeting ligand, and for which a retargeting ligand, e.g., antibodies or portions thereof, are already available. Such structures include, but are not limited to: the major histocompatibility class I and class II antigens; receptors for a variety of cytokines (e.g., receptors for IL-1, IL-4, IL-6, IL-13, IL-22, IL-25, IL-33, etc.), cell-type-specific growth hormones, brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CTNF), colony-stimulating growth factors, endothelial growth factors, epidermal growth factors, fibroblast growth factors, glial cell-derived neurotrophic factor, glial growth factors, gro-beta/mip 2, hepatocyte growth factors, insulin-like growth factor, interferons (a-IFN, p-IFN, and IFN, consensus IFN), interleukins (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14), keratinocyte growth factor, leukemia inhibitory factors, macrophage/monocyte chemotactic activating factor, nerve growth factor, neutrophil-activating protein 2, platelet-derived growth factor, stem cell factor, transforming growth factor, tumor necrosis factors, vascular endothelial growth factor, lipoproteins, including additional or other type 1 transmembrane receptors such as PRLR, G protein-coupled receptors such as GCGR, ion channels such as Nav1.7, ASIC1, or ASIC2; cell adhesion molecules; transport molecules for metabolites such as amino acids; B and T cell antigen receptors (e.g., B cell receptors and associated proteins (e.g., CD19, CD20, etc.) and T cell receptors and associated proteins (e.g., CD3, CD4, CD8, etc.); a tetraspanin protein (e.g., CD63). A recombinant AAV capsid described herein enables cell type-specific infection by employing a multispecific binding molecule comprising a homing ligand that binds to differentiation cell surface antigens as targets for the AAV vector complex.

In some embodiments, the retargeting ligand binds to a protein expressed primarily (e.g., only) by (human) liver cells, i.e., a liver-specific marker. In some embodiments, the retargeting ligand binds to a protein expressed primarily (e.g., only) by (human) brain cells, a brain cell-specific marker. In some embodiments, the retargeting ligand binds to a protein expressed primarily (e.g., only) by (human) hematopoietic cells, i.e., a hematopoietic cell-specific marker. In some embodiments, the retargeting ligand binds to a protein expressed primarily (e.g., only) by (human) T cells, i.e., a T cell-specific marker. In some embodiments, the retargeting ligand binds to a protein expressed primarily (e.g., only) by (human) B cells, i.e., a B cell-specific marker. In some embodiments, the retargeting ligand binds to a protein expressed primarily (e.g., only) by (human) dendritic cells, i.e., a dendritic cell-specific marker. In some embodiments, the retargeting ligand binds to a protein expressed primarily (e.g., only) by (human) macrophages, i.e., a macrophage-specific marker. In some embodiments, the retargeting ligand binds to a protein expressed primarily (e.g., only) by (human) NK cells, i.e., an NK cell-specific marker. In some embodiments, the retargeting ligand binds to a protein expressed primarily (e.g., only) by (human) kidney cells, i.e., a kidney-specific marker. In some embodiments, the retargeting ligand binds to a receptor expressed primarily (e.g., only) by (human) pancreas cells, i.e., a pancreas-specific marker. In some embodiments, the retargeting ligand binds to a receptor expressed primarily (e.g., only) by (human) intestinal cells, i.e., a gut-specific marker. In some embodiments, the retargeting ligand binds to a protein expressed primarily (e.g., only) by a (human) cancer cell, i.e., a tumor-associated antigen. In some embodiments, the retargeting ligand binds to a protein expressed primarily (e.g., only) by (human) cells infected with a heterologous pathogen. Proteins that are (1) specifically expressed, or whose expression is enriched in, a cell/tissue/organ, and (2) recognized by an antigen-binding protein useful as a retargeting ligand as described herein are well known and can also be found at www.proteinatlas.org; see also Uhlenet al.(2010)Nat. Biotech.

28:1248-50. Table 1 below provides illustrative and non-limiting organ-specific markers for which antigen-binding proteins, which may be useful as retargeting ligands, are available and the cells/tissues/organs that express such markers.

T l 1: M r r ífi i il r iv

In some embodiments, the retargeting ligand binds to a receptor expressed by a (human) liver cell, e.g., an asialoglycoprotein receptor, e.g., hASGR1. In some embodiments, the retargeting ligand binds to a receptor expressed by a (human) brain cell. In some embodiments, the retargeting ligand binds to a receptor expressed by a (human) T cell, e.g., CD3, e.g., CD3e. In some embodiments, the retargeting ligand binds to a receptor expressed by a (human) kidney cell. In some embodiments, the retargeting ligand binds to a receptor expressed by a (human) muscle cell, e.g., an integrin. In some embodiments, the retargeting ligand binds to a receptor expressed by a (human) cancer cell, e.g., a tumor-associated antigen, e.g., E6 and E7. In some embodiments, the retargeting ligand binds to the human glucagon receptor (hGCGR). In some embodiments, the retargeting ligand binds to human ENTPD3.

In some embodiments, the retargeting ligand binds to a tumor-associated antigen expressed by a tumor cell. Non-limiting examples of specific tumor associated antigens include, for example, adipophilin, AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein (A<f>P), ARTC1, B-RAF, BAGE-1, BCLX(L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen (CEA), CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin D1, Cyclin-A1, dek-can fusion protein, DKK1, EFTUD2, Elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen (ETA), ETV6-AML1 fusion protein, EZH2, E6,<e>7, FGF5, FLT3-ITD, FN1, G250/MN/CAIX, GAGE-1,2,8, GAGE-3,4,5,6,7, GAS7, glypican-3, GnTV, gp100/Pme117, GPNMB, HAUS3, Hepsin, 2/neu, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3, IL13Ralfa2, Intestinal carboxyl esterase, K-ras, Kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1 aka CCDC110, LAGE-1, fusion protein LDLR-fucosyltransferaseAS, Lengsin, M-CSF, MAGE-A1, MAGE-A 10, MAGE-A12, MAGE-A2, Ma Ge -A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malic enzyme, mammaglobin-A, MART2, MATN, MC1R, MCSP, mdm-2, ME1/ MART-1, Meloe, Midquina, MMP-2, MMP-7, MUC1, MUC5AC, mucin, MUM-1, MUM-2, MUM-3, Myosin, Myosin class I, N-raw, NA88-A, neo-PAP, NFYC, NY-BR-1, NYESO-1/LAGE-2, OA1, OGT, OS-9, P polypeptide, p53, PAP, PAX5, PBF, pml-RARalpha fusion protein, polymorphic epithelial mucin (PEM), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38/NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, secernin 1, SIRT2, SNRPD1, SOX10, Sp17, 17, SSX-2, SSX-4, STEAP1, survivin, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2, Telomerase, TGF-betaRII, TPBG, TRAG-3, Triosephosphate isomerase, TRP-1/gp75, TRP-2, TRP2-INT2, tyrosinase, tyrosinase (TYR), VEGF, WT1, XAGE-lb/GAGED2a, Kras, NY-ESO1, MAGE-A3, HPV E2, HPV E6, HPV E7, WT antigen-1 (in lymphoma and other solid tumors), ErbB receptors, Melan A [MART1], gp 100, tyrosinase, TRP-1/gp 75, and TRP-2 (in melanoma); MAGE-1 and MAGE-3 (in bladder, head and neck, and non-small cell carcinoma); HPV EG and E7 proteins (in cervical cancer); Mucin [MUC-1] (in breast, pancreas, colon, and prostate cancers); prostate-specific antigen [PSA] (in prostate cancer); carcinoembryonic antigen [CEA] (in colon, breast, and gastrointestinal cancers) and shared tumor-specific antigens such as MAGE-2, MAGE-4, MAGE-6, MAGE-10, MAGE-12, BAGE-1, CAGE-1,2,8, CAGE-3 TO 7, LAGE-1, NY-ESO-1/LAGE-2, NA-88, GnTV, TRP2-INT2. In some embodiments, the tumor associated antigen is ErbB2/Her2. In some embodiments, the tumor associated antigen is E6 and/or E7.

In some embodiments, the retargeting ligand binds to CD markers associated with immune response, e.g., CD3, CD4, CD8, CD19, CD20, etc. In some embodiments, the CD marker is CD3.

In certain illustrative embodiments, the multispecific binding molecule is a bispecific antibody. Each antigen-binding domain of a bispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR). In the context of a bispecific antigen-binding molecule comprising a first and a second antigen-binding domain (e.g., a bispecific antibody), the CDRs of the first antigen-binding domain may be designated with the prefix "A1" and the CDRs of the second antigen-binding domain may be designated with the prefix "A2." Thus, the CDRs of the first antigen-binding domain may be referred to herein as A1-HCDR1, A1-HCDR2, and A1-HCDR3; and the CDRs of the second antigen-binding domain may be referred to herein as A2-HCDR1, A2-HCDR2, and A2-HCDR3.

The first antigen binding domain and the second antigen binding domain may be directly or indirectly connected to each other to form a bispecific antigen binding molecule of the present invention. Alternatively, the first antigen binding domain and the second antigen binding domain may be linked to a separate multimerization domain. Association of one multimerization domain with another multimerization domain facilitates association between the two antigen binding domains, thereby forming a bispecific antigen binding molecule. As used herein, a "multimerization domain" is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to associate with a second multimerization domain of the same or similar structure or constitution. For example, a multimerizing domain may be a polypeptide comprising an immunoglobulin CH3 domain. A non-limiting example of a multimerization component is an Fc portion of an immunoglobulin (comprising a CH2-CH3 domain), for example, an Fc domain of an IgG selected from the isotypes IgG1, IgG2, IgG3 and IgG4, as well as any allotypes within each isotype group.

The bispecific antigen binding molecules employed in the present invention will typically comprise two multimerization domains, for example, two Fc domains that are each individually part of a separate antibody heavy chain. The first and second multimerization domains may be of the same IgG isotype such as, for example, IgG1/IgG1, IgG2/IgG2, IgG4/IgG4. Alternatively, the first and second multimerization domains may be of different IgG isotypes such as, for example, IgG1/IgG2, IgG1/IgG4, IgG2/IgG4, etc.

In certain embodiments, the multimerization domain is an Fc fragment or an amino acid sequence 1 to about 200 amino acids in length containing at least one cysteine residue. In other embodiments, the multimerization domain is a cysteine residue or a short cysteine-containing peptide. Other multimerization domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix loop motif, or a coiled-coil motif.

Any bispecific antibody format or technology can be used to produce the bispecific antigen-binding molecules employed in the present invention. For example, an antibody or fragment thereof having a first antigen-binding specificity can be operably linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity to produce a bispecific antigen-binding molecule. Other specific illustrative bispecific formats that can be used in the context of the present invention include, without limitation, for example, scFv-based or diabody bispecific formats, IgG-scFv fusions, double variable domain (DVD)-Ig, quadrome, "buttonhole", common light chain (e.g., common light chain with "buttonhole", etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1/IgG2, dual-acting Fab (DAF)-IgG, and Mab2 bispecific formats (see, e.g., Klein et al., 2012, mAbs4:6, 1-11, and references cited therein, for a review of the above formats; see also Brinkmann and Conterman (2017) mAbs9:182-212.

The present invention may also employ bispecific antigen binding molecules comprising a first CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from each other by at least one amino acid, and wherein the at least one amino acid difference reduces binding of the bispecific antibody to protein A as compared to a bispecific antibody lacking the amino acid difference. In one embodiment, the first Ig CH3 domain binds protein A and the second Ig CH3 domain contains a mutation that reduces or abolishes protein A binding such as an H95R modification (according to IMGT exon numbering; H-435-R according to EU numbering). The second CH3 may further comprise a Y96F modification (according to IMGT; Y436F according to EU). Other modifications that can be found in the second CH3 include: D-16-E, L-18-M, N-44-S, K-52-N, V-57-M, and V-82-I (according to IMGT; D-356-E, L-358-M, N-384-S, K-392-N, V-397-M, and V-422-I according to EU) in the case of IgG1 antibodies; N-44-S, K-52-N, and V-82-I (IMGT; N-384-S, K-392-N, and V-422-I according to EU) in the case of IgG2 antibodies; and Q-15-R, N-44-S, K-52-N, V-57-M, R-69-K, E-79-Q and V-82-I (according to IMGT; Q-355-R, N-384-S, K-392-N, V-397-M, R-409-K, E-419-Q and V-422-I according to EU) in the case of IgG4 antibodies; see, for example, WO 2010/151792.

In certain embodiments, the Fc domain may be chimeric, combining Fc sequences from more than one immunoglobulin isotype. For example, a chimeric Fc domain may comprise part or all of a CH2 sequence derived from a CH2 region of a human IgG1, a human IgG2, or a human IgG4, and part or all of a CH3 sequence derived from a human IgG1, a human IgG2, or a human IgG4. A chimeric Fc domain may also contain a chimeric hinge region. For example, a chimeric hinge may comprise an "upper hinge" sequence, derived from a hinge region of a human IgG1, a human IgG2, or a human IgG4, together with a "lower hinge" sequence, derived from a hinge region of a human IgG1, a human IgG2, or a human IgG4. A particular example of a chimeric Fc domain that can be included in any of the antigen binding molecules set forth herein comprises, from N-terminus to C-terminus: [CH1 of IgG4]-[upper hinge of IgG4]-[lower hinge of IgG2]-[CH2 of IgG4]-[CH3 of IgG4]. Another example of a chimeric Fc domain that can be included in any of the antigen binding molecules set forth herein comprises, from N-terminus to C-terminus: [CH1 of IgG1]-[upper hinge of IgG1]-[lower hinge of IgG2]-[CH2 of IgG4]-[CH3 of IgG1]. These and other examples of chimeric Fc domains that can be included in any of the antigen binding molecules of the present invention are described in PCT application No. WO2014/022540. Chimeric Fc domains that have these general structural arrangements and variants thereof may have altered Fc receptor binding, which in turn affects Fc effector function.

Methods of use and production

A further embodiment of the recombinant AAV capsid proteins described herein is their use to deliver a nucleotide of interest, e.g., a reporter gene or a therapeutic gene, to a target cell. Thus, the present invention provides a composition of the present invention for use in a method of treatment or diagnosis in vivo, the method comprising administering the nucleotide of interest to a target cell expressing a cell surface protein comprising contacting the target cell with the composition, wherein the multispecific binding molecule comprises a retargeting ligand that binds to the cell surface protein. The present invention further provides a method for administering a nucleotide of interest in vitro or ex-vivo to a target cell expressing a cell surface protein comprising contacting the target cell with the composition of the present invention, wherein the multispecific binding molecule comprises a retargeting ligand that binds to the cell surface protein.

Generally, a nucleotide of interest may be a transfer plasmid, which may typically comprise 5' and 3' inverted terminal repeat (ITR) sequences flanking the reporter gene(s) or therapeutic gene(s) (which may be under the control of a viral or non-viral promoter, when included within an AAV vector). In one embodiment, a nucleotide of interest is a transfer plasmid comprising from 5' to 3': a 5' ITR, a promoter, a gene (e.g., a reporter and/or therapeutic gene), and a 3' ITR.

Useful non-limiting examples of promoters include, for example, the cytomegalovirus (CMV) promoter, the spleen focus forming virus (SFFV) promoter, the elongation factor 1 alpha (EF1a) promoter (either the 1.2 kb EF1a promoter or the 0.2 kb EF1a promoter), the EF1a/IF4 chimeric promoter, and the phosphoglycerate kinase (PGK) promoter. An internal enhancer may also be present in the viral construct to increase expression of the gene of interest. For example, the CMV enhancer (Karasuyama et al. 1989. J. Exp. Med. 169:13) may be used. In some embodiments, the CMV enhancer may be used in conjunction with the chicken actin promoter.

A variety of reporter genes (or detectable moieties) can be encapsulated into a multimeric structure comprising the recombinant AAV capsid proteins described herein. Illustrative reporter genes include, for example, p-galactosidase (encoded genlacZ), Green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), MmGFP, blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, Yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), Emerald, CyPet, Cyan fluorescent protein (CFP), Cerulean, T-Sapphire, Luciferase, Alkaline phosphatase, or a combination thereof. The methods described herein demonstrate the construction of targeting vectors employing the use of a reporter gene encoding green fluorescent protein, however, those of skill reading this disclosure will understand that the non-human animals described herein may be generated in the absence of a reporter gene or with any reporter gene known in the art.

A variety of therapeutic genes can also be encapsulated in a multimeric structure comprising the recombinant AAV capsid proteins described herein, for example, as part of a transfer vector. Non-limiting examples of a therapeutic gene include those encoding a toxin (e.g., a suicide gene), a therapeutic antibody or fragment thereof, a CRISPR/Cas system or portion(s) thereof, antisense RNA, siRNA, shRNA, etc.

A further embodiment of the present invention is a process for the preparation of a recombinant AAV capsid protein, the method comprising the steps of:

a) express a nucleic acid encoding the recombinant AAV capsid protein under suitable conditions, and

b) isolate the capsid protein expressed from step a).

Other embodiments of the present invention are a method for altering the tropism of a virus, the method comprising the steps of: (a) inserting a nucleic acid encoding a heterologous epitope into a nucleic acid sequence encoding an AAV capsid protein to form a nucleotide sequence encoding a genetically modified AAV capsid protein comprising the heterologous epitope and/or (b) culturing a packaging cell under conditions sufficient for the production of AAV vectors, wherein the packaging cell comprises the nucleotide sequence. A further embodiment of the present invention is a method for presenting a heterologous epitope on the surface of an AAV capsid protein, the method comprising the steps of: a) expressing the nucleic acid according to this invention under suitable conditions, and b) isolating the AAV capsid protein expressed in step a).

In some embodiments, the packaging cell further comprises a helper plasmid and/or a transfer plasmid comprising a nucleotide of interest. In some embodiments, the methods further comprise isolating self-complementary adeno-associated viral vectors from the culture supernatant. In some embodiments, the methods further comprise lysing the packaging cell and isolating single-stranded adeno-associated viral vectors from the cell lysate. In some embodiments, the methods further comprise (a) removing cellular debris, (b) treating the supernatant containing AAV vectors with DNase I and MgCl2, (c) concentrating the viral vectors, (d) purifying the viral vectors, and (e) any combination of (a)-(d).

Packaging cells useful for the production of the AAV vectors described herein include, for example, animal cells permissive for the virus, or cells modified to be permissive for the virus; or the construction of packaging cells, for example, with the use of a transformation agent such as calcium phosphate. Non-limiting examples of packaging cell lines useful for producing AAV vectors described herein include, for example, human embryonic kidney 293 (HEK-293) cells (e.g., American Type Culture Collection [ATCC] No. CRL-1573), HEK-293 cells containing the SV40 large T antigen (HEK-293T or 293T), HEK293T/17 cells, human sarcoma cell line HT-1080 (CCL-121), Raj i lymphoblast-like cell line (CCL-86), glioblastoma-astrocytoma epithelial-like cell line U87-MG (HTB-14), HuT78 T-lymphoma cell line (TIB-161), NIH/3T3 cells, Chinese hamster ovary (CHO) cells (e.g., ATCC No. CRL9618, CCL61, CRL9096), HeLa cells (e.g., ATCC No. CCL-2), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, Hk b cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), HLHepG2 cells, CAP cells, CAP-T cells and the like.

L929 cells, the FLY viral packaging cell system described in Cosset et al (1995) J Virol 69, 7430-7436, NS0 cells (murine myeloma), human amniocytic cells (e.g. CAP, CAP-T), yeast cells (including but not limited to S. cerevisiae, Pichia shepherdis), plant cells (including but not limited to Tobacco NT1, BY-2), insect cells (including but not limited to SF9, S2, SF21, Tni (e.g. High 5)) or bacterial cells (including but not limited to E. coli).

For additional packaging cells and systems, packaging techniques, and vectors for packaging the nucleic acid genome into the pseudotyped viral vector see, for example, Polo, et al, Proc Natl Acad Sci USA, (1999) 96:4598-4603. Packaging methods include the use of packaging cells that permanently express the viral components or by transient transfection of cells with plasmids.

Additional embodiments include methods for redirecting an AAV virus and/or delivering a reporter or therapeutic gene to a target cell, the method comprising a method for transducing cells in vitro, the method comprising the steps of: contacting the target cell with the combination of an AAV vector comprising a capsid comprising a recombinant AAV capsid displaying a heterologous epitope and a multispecific binding molecule, wherein the multispecific binding molecule comprises (i) an antibody paratope that specifically binds to the epitope and (ii) a redirection ligand that specifically binds to a receptor expressed by the target cell. In some embodiments, a composition described herein comprises, or a method described herein combines, a recombinant AAV vector and a multispecific binding molecule in a molecule-molecule ratio that restores the transduction efficiency of the AAV vector similar to that of a wild-type control AAV vector. In some embodiments, the ratio of recombinant AAV vector to multispecific binding molecule (molecule:molecule) ranges from 1:0.5 to 1:100. In some embodiments, the ratio of recombinant AAV vector to multispecific binding molecule (molecule:molecule) ranges from 1:4 to 1:20. In some embodiments, the ratio of recombinant AAV vector to multispecific binding molecule (molecule:molecule) ranges from 1:8 to 1:15. In some embodiments, the ratio of recombinant AAV vector to multispecific binding molecule (molecule:molecule) is 1:4. In some embodiments, the ratio of recombinant AAV vector to multispecific binding molecule (molecule:molecule) is 1:8. In some embodiments, the ratio of recombinant AAV vector to multispecific binding molecule (molecule:molecule) is 1:15. In some embodiments, the ratio of recombinant AAV vector to multispecific binding molecule (molecule:molecule) is 1:20. In some embodiments, the ratio of recombinant AAV vector to multispecific binding molecule (molecule:molecule) is less than 1:100. In some embodiments, the ratio of recombinant AAV vector to multispecific binding molecule (molecule:molecule) is less than 1:50. In some embodiments, the ratio of recombinant AAV vector to multispecific binding molecule (molecule:molecule) is less than 1:20. In some embodiments, the ratio of recombinant AAV vector to multispecific binding molecule (molecule:molecule) is less than 1:15. In some embodiments, the ratio of recombinant AAV vector to multispecific binding molecule (molecule:molecule) is less than 1:10.

In some embodiments, the target cell is in vitro. In other embodiments, the invention provides a composition for use in a method of treatment or diagnosis in vivo, the method comprising administering the nucleotide of interest to a target cell expressing a cell surface protein, comprising contacting the target cell with the composition. Thus, in such embodiments the target cell is in vivo in a subject, e.g., a human.

Target cells

A wide variety of cells can be targeted to deliver a nucleotide of interest using a recombinant AAV vector as described herein. Target cells will generally be chosen based on the nucleotide of interest and the desired effect.

In some embodiments, a nucleotide of interest can be administered to enable a target cell to produce a protein that compensates for a deficiency in an organism, such as an enzyme deficiency or immunodeficiency, such as X-linked severe combined immunodeficiency. Thus, in some embodiments, cells that would normally produce the protein in the animal are targeted. In other embodiments, cells in the area where a protein would be most beneficial are targeted.

In other embodiments, a nucleotide of interest, such as a gene encoding an siRNA, can inhibit the expression of a particular gene in a target cell. The nucleotide of interest can, for example, inhibit the expression of a gene involved in the life cycle of a pathogen. Thus, cells susceptible to infection by the pathogen or infected with the pathogen can be targeted. In other embodiments, a nucleotide of interest can inhibit the expression of a gene responsible for the production of a toxin in a target cell.

In other embodiments, a nucleotide of interest may encode a toxic protein that kills the cells in which it is expressed. In this case, tumor cells or other unwanted cells may be targeted.

In still other embodiments, a nucleotide of interest encoding a protein to be harvested, such as a therapeutic protein, can be used and directed to cells that are capable of producing and secreting the protein.

Once a particular target cell population is identified in which expression of a nucleotide of interest is desired, a target receptor is selected that is specifically expressed in that target cell population. The target receptor may be expressed exclusively in that cell population or to a greater extent in that cell population than in other cell populations. The more specific the expression, the more specifically the target cells can be targeted. Depending on the context, the desired amount of specificity of the marker (and therefore of the gene delivery) may vary. For example, for introduction of a toxic gene, high specificity is most preferred to avoid killing non-target cells. For expression of a protein for harvesting, or expression of a secreted product where global impact is desired, lower marker specificity may be necessary.

As discussed above, the target receptor can be any receptor for which a retargeting ligand can be identified or created. Preferably the target receptor is a peptide or polypeptide, such as a receptor. However, in other embodiments, the target receptor can be a carbohydrate or other molecule that can be recognized by a binding partner. If a binding partner, e.g., ligand, for the target receptor is already known, it can be used as an affinity molecule. However, if a binding molecule is not known, antibodies against the target receptor can be generated using conventional methods. The antibodies can then be used as the retargeting ligand as part of the multispecific binding molecule.

Target cells can therefore be chosen based on a variety of factors, including, for example, (1) the particular application (e.g., therapy, expression of a protein to be harvested and conferring disease resistance) and (2) expression of a marker with the desired amount of specificity.

Target cells are not limited in any way and include both germline cells and cell lines and somatic cells and cell lines. Target cells may be stem cells derived from any origin. When target cells are germline cells, the target cells are preferably selected from the group consisting of single-cell embryos and embryonic stem (ES) cells.

Pharmaceutical compositions, pharmaceutical forms and administration

A further embodiment provides a medicament comprising at least one recombinant AAV capsid protein and an appropriate multispecific binding molecule according to this invention and/or a nucleic acid according to this invention, preferably at least one multimeric structure according to this invention. Preferably said medicament is useful as a gene transfer vector.

Also described herein are pharmaceutical compositions comprising the AAV vectors described herein and a pharmaceutically acceptable carrier and/or excipient. Additionally, disclosed herein are dosage forms comprising the AAV vector described herein.

As discussed herein, the AAV vectors described herein can be used for various therapeutic applications (in vivo and ex-vivo) and as research tools.

Pharmaceutical compositions based on the AAV vectors disclosed herein may be formulated in any conventional manner using one or more physiologically acceptable carriers and/or excipients. The AAV vector may be formulated for administration by, for example, injection, inhalation or isolation (either via the mouth or nose) or by oral, buccal, parenteral or rectal administration, or by administration directly to a tumor.

Pharmaceutical compositions may be formulated for a variety of modes of administration, including systemic, topical, or local administration. Techniques and formulations may be found in, for example, Remington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For the purpose of injection, pharmaceutical compositions may be formulated in liquid solutions, preferably in physiologically compatible buffers, such as Hank's solution or Ringer's solution. In addition, pharmaceutical compositions may be formulated in solid form and re-dissolved or suspended immediately prior to use. Lyophilized forms of the pharmaceutical composition are also suitable.

For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavorings, colorings, and sweetening agents, as appropriate.

The pharmaceutical compositions may be formulated for parenteral administration by injection, for example, by bolus or continuous infusion. Formulations for injection may be presented in unit dosage form, for example, in ampoules or in multidose containers, with an optionally added preservative. The pharmaceutical compositions may also be formulated as suspensions, solutions or emulsions in oleaginous or aqueous vehicles and may contain other agents including suspending, stabilizing and/or dispersing agents.

Additionally, the pharmaceutical compositions may also be formulated as a slow release ("depot") preparation. Such long acting formulations may be administered by implant (e.g. subcutaneous or intramuscular) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (e.g. as an emulsion in acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g. as a sparingly soluble salt. Other suitable delivery systems include microspheres, which offer the possibility of noninvasive local drug delivery over an extended period of time. This technology may include microspheres having a precapillary size, which can be injected through a coronary catheter into any selected part of an organ without causing inflammation or ischemia. The delivered therapeutic agent is then slowly released from the microspheres and taken up by surrounding cells present in the selected tissue.

Systemic administration may also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration may be accomplished by nasal sprays or suppositories. For topical administration, the AAV vector described herein may be formulated into ointments, balms, gels, or creams, as is generally known in the art. A wash solution may also be used locally to treat an injury or inflammation to accelerate healing.

Pharmaceutical forms suitable for injectable use may include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid. It must be stable under the conditions of manufacture and certain storage parameters (e.g., refrigeration and freezing) and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

If the formulations disclosed herein are used as a therapeutic to stimulate an immune response in a subject, a therapeutic agent may be formulated in a composition in a neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids or such organic acids as acetic, oxalic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups may also be obtained from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like.

A carrier may also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils. Suitable fluidity may be maintained, for example, by using a coating, such as lecithin, by maintaining the required viral vector size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents known in the art. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions may be brought about by using absorption delaying agents in the compositions, for example, aluminum monostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating the active compounds or constructs in the required amount in the appropriate solvent with other ingredients listed above as necessary, followed by sterilization by filtration.

After formulation, the solutions can be administered in a manner compatible with the pharmaceutical formulation and in an amount such that it is therapeutically effective. The formulations are readily administered in a variety of pharmaceutical forms, such as the type of injectable solutions described above, but slow-release capsules or microparticles and microspheres and the like may also be employed.

For parenteral administration in an aqueous solution, for example, the solution must be suitably buffered if necessary and the liquid diluent must first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intratumoral, intramuscular, subcutaneous and intraperitoneal administration. In this context, sterile aqueous media which may be employed will be known to those skilled in the art in light of the present disclosure. For example, a dose could be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of hypodermolysis fluid or injected into the proposed infusion site.

The person responsible for administration will, in any case, determine the appropriate dosage for the individual subject. For example, a subject may be administered the AAV vectors described herein on a daily or weekly basis for a period of time or on a monthly, biennially, or annually basis depending on need or exposure to a pathogen or condition in the subject (e.g., cancer).

In addition to compounds formulated for parenteral administration, such as intravenous, intratumoral, intradermal or intramuscular injection, other pharmaceutically acceptable forms include, for example, tablets or other solids for oral administration; liposomal formulations; controlled release capsules; biodegradable and any other form currently used.

Intranasal or inhalable solutions or aerosols, sprays or inhalants, may also be used. Nasal solutions may be aqueous solutions designed to be administered to the nasal passages in drops or sprays. Nasal solutions may be prepared so as to be similar in many respects to nasal secretions. Thus, aqueous nasal solutions are usually isotonic and slightly buffered to maintain a pH of 5.5 to 7.5. In addition, antimicrobial preservatives, similar to those used in ophthalmic preparations, and appropriate drug stabilizers, if necessary, may be included in the formulation. Various commercial nasal preparations are known, and may include, for example, antibiotics and antihistamines and are used for asthma prophylaxis.

Oral formulations may include excipients such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders. In certain defined embodiments, oral pharmaceutical compositions will include an inert diluent or assimilable edible carrier, or may be enclosed in a hard or soft gelatin capsule, or may be compressed into tablets, or may be incorporated directly into the food of the diet. For oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like.

Tablets, troches, pills, capsules and the like may also contain the following: a binder, such as gum tragacanth, acacia, cornstarch or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavor, may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or otherwise to modify the physical form of the dosage unit. For example, tablets, pills or capsules may be coated with shellac, sugar or both. An elixir syrup may contain the active compounds sucrose as a sweetening agent, methyl and propyl parabens as preservatives, a colorant, and a flavoring, such as cherry or orange flavor.

Other embodiments disclosed herein may relate to kits for use with methods and compositions. Kits may also include a suitable container, for example, vials, tubes, mini- or microcentrifuge tubes, test tube, flask, bottle, syringe, or other container. Where an additional component or agent is provided, the kit may contain one or more additional containers into which this agent or component may be placed. The kits herein will typically also include a means for containing the AAV vectors and any other reagent containers in closed confinement for commercial sale. Such containers may include injected or blow-molded plastic containers in which the desired vials are held. Optionally, one or more additional active ingredients such as, for example, anti-inflammatory agents, antiviral agents, antifungal or antibacterial agents, or antitumor agents may be required for the disclosed compositions.

Dosage ranges and frequency of administration may vary depending on the nature of the AAV vectors and the medical condition, as well as the parameters of a specific patient and the route of administration used. In some embodiments, AAV vector compositions may be administered to a subject at a dose ranging from about 1x10 plaque forming units (pfu) to about 1x10 pfu, depending on the mode of administration, route of administration, nature of the disease, and condition of the subject. In some cases, AAV vector compositions may be administered at a dose ranging from about 1x10 pfu to about 1x10 pfu, or from about 1x10 pfu to about 1x10 pfu, or from about 1x10 pfu to about 1x10 pfu. A more precise dose may also depend on the subject to which it is administered. For example, a lower dose may be necessary if the subject is juvenile, and a higher dose may be necessary if the subject is an adult human subject. In certain embodiments, a more precise dose may depend on the weight of the subject. In certain embodiments, for example, a juvenile human subject may receive from about 1x108 pfu to about 1x1010 pfu, while an adult human subject may receive a dose of from about 1x1010 pfu to about 1x1012 pfu.

The compositions disclosed herein can be administered by any means known in the art. For example, the compositions can include administration to a subject intravenously, intratumorally, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intrathecally, subcutaneously, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion, by a catheter, by a lavage, in a cream, or in a lipid composition.

Any method known to one of skill in the art can be used for the large scale production of AAV vectors, packaging cells, and vector constructs described herein. For example, master and working seed stocks can be prepared under GMP conditions in qualified primary CEFs or by other methods. Packaging cells can be seeded into large surface area flasks, grown to near confluence, and AAV vectors purified. Cells can be harvested and AAV vectors released into the isolated and purified culture media, or intracellular AAV vectors can be released by mechanical disruption (cellular debris can be removed by large pore depth filtration and host cell DNA can be digested with endonuclease). AAV virus vectors can be further purified and concentrated by tangential flow filtration, followed by diafiltration. The resulting concentrated mass can be formulated by dilution with a buffer containing stabilizers, packaged into vials, and lyophilized. Compositions and formulations can be stored for later use. For use, lyophilized AAV vectors can be reconstituted by the addition of diluent.

Certain additional agents used in combination therapies may be formulated and administered by any means known in the art.

The compositions as disclosed herein may also include adjuvants such as aluminum salts and other mineral adjuvants, surface active agents, bacterial derivatives, carriers, and cytokines. Adjuvants may also have antagonistic immunomodulatory properties. For example, adjuvants may stimulate Th1 or Th2 immunity. The compositions and methods disclosed herein may also include adjuvant therapy.

Examples

The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1: Production of adeno-associated viral vectors with a heterologous epitope

AAV capsid proteins are modified to contain one of several heterologous epitopes: FLAG, c-myc, hexahistidine, etc. using PCR to generate a plasmid encoding a recombinant capsid protein. Briefly, the sequence encoding FLAG, c-myc, or hexahistidine is inserted in frame after the codon encoding N587 of an AAV2 capsid protein, Q585 of an AAV6 VP1 capsid protein, N590 of an AAV8 VP1 capsid protein, A589 of an AAV9 VP1 capsid protein, or G453 of an AAV9 VP1 capsid protein.

Adeno-associated virus production is performed by a triple transfection method with HEK293 cells (see for example Erik Arden and Joseph M. Metzger, J. Biol. Methods. 2016; 3(2)). Cells are plated one day prior to PEFpro-mediated transfection (Polyplus Transfection, New York, NY) with appropriate vectors:

A helper plasmid, pHelper (Agilent, cat. no. 240074);

A plasmid encoding the wild-type or modified AAV gene p/cap (pAAV RC2 (Cell Biolabs, cat. no. VPK-422), e.g., pAAV RC2/6/9 (Cell Biolabs, cat. no. VPK-426), pAAV RC8, pAAV RC2-N587myc, pAAV RC2/6-Q585myc, pAAVRC8-N590myc, pAAV RC9-A589myc, etc.; and

A plasmid encoding a nucleotide of interest and AAV ITR sequences, e.g., pscAAV-CMV-eGFP, pAAV-CMVGFP (Agilent Cat. No. 240074), pAAV-EF1a-eGFP, or pAAV-CAGG-eGFP, etc.

Seventy-two hours after transfection, the medium is harvested and cells are lysed in buffer [50 mM Tris-HCl, 150 mM NaCl, 0.5% sodium deoxycholate (Sigma, cat. no. D6750-100G)]. Benzonase (Sigma, St. Louis, MO) is then added to both the medium and cell lysate to a final concentration of 0.5 U/pl before incubation at 37°C for 60 minutes. The cell lysate is centrifuged at 4,000 rpm for 30 minutes. The cell lysate and medium are combined and precipitated with PEG 8000 (Teknova cat. no. P4340) to a final concentration of 8%. The pellet is resuspended in 400 mM NaCl and centrifuged at 10,000 g for 10 minutes. Viruses in the supernatant are pelleted by ultracentrifugation at 149,000 g for 3 hours and titrated by qPCR.

For qPCR to titrate AAV genomes, AAV samples are treated with DNAsalt (Thermofisher Scientific, cat. no. EN0525) at 37°C for one hour and lysed using all DNA extract reagents (Thermofisher Scientific cat. no. 4403319). Encapsidated viral genomes are quantified using a QuantStudio 3 Real-Time PCR System (Thermofisher Scientific) using primers targeting the AAV2 ITRs. The AAV2 ITR primer sequences are 5-GGAACCCCTAGTGATGGAGTT-3' (forward ITR; SEQ ID NO:9) and 5'-CGGCCTCAGTGAGCGA-3' (reverse ITR; SEQ ID NO: 10) (Aurnhammer et al.,2012), derived from the left internal inverted repeat (ITR) sequence of AAV and the right internal inverted repeat (ITR) sequence of <a>A<v>, respectively. The AAV2 ITR probe sequence is 5'-6-FAM-CACTCCCTCT<g>C<g>CGCTCG-TAMRA-3' (SEQ ID NO:11) (Aurnhammer C., Haase M., Muether N.,et al.,2012,Hum. Gene Ther. Methods23, 18 28). After an activation step at 95°C for 10 min, a two-step PCR cycle is performed at 95°C for 15 s and 60°C for 30 s for 40 cycles. TaqMan Universal PCR Master Mix (Thermofisher Scientific, cat. no. 4304437) was used for qPCR. Plasmid DNA (Agilent, cat. no. 240074) is used as a standard to determine absolute titers.

Adeno-associated viral vectors comprising a capsid in which a c-myc epitope has been produced. In this example, a c-myc epitope (EQKLISEEDL; SEQ ID NO:6) was inserted between amino acids N587 and R588 of the AAV2 VP1 capsid protein or between amino acids A589 and Q590 of the AAV9 VP1 capsid protein, i.e., the nucleotide sequence encoding the c-myc epitope (GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG; SEQ ID NO:12) was inserted into plasmid pAAV RC2 (Cell Biolabs, Inc., San Diego, CA) or plasmid pAAV RC2/9, and each of the respective modified pAAV RC2-N587Myc and pAAV RC9-A589Myc plasmids were used to encode the modified capsid protein for AAV viral vectors with a reduced or null tropism.

To create pAAV RC2-N587Myc, a first polymerase chain reaction (PCR) product comprising (5' to 3') a BsiW1 restriction site, the nucleotide sequence between positions 3050 and 3773 of pAAV RC2, and a nucleotide sequence overhanging the c-myc epitope, and a second PCR product comprising (5' to 3') a nucleotide sequence overhanging the c-myc epitope, the nucleotide sequence between positions 3774 to 4370 of pAAV RC2, and a Pme1 restriction site were created using the primers set forth in Table 2. Plasmid pAAV RC2-N587Myc (i.e., a pAAV RC2 plasmid modified to encode the c-myc epitope between amino acids N587 and R588 of the protein) was created using the primers set forth in Table 2. of the VP1 capsid) was created by digesting pAAV RC2 with BsiW1 (New England Biolabs, R0553L) and Pme1 (New England Biolabs, R0560L), and inserting the two PCR products by ligation-independent cloning as described in (2012) Methods Mol. Biol. 52:51–9.

To create pAAV RC2/6-Q585Myc, a gblock DNA fragment comprising positions 3700 and 3940 of pAAV RC2/6 with the c-myc epitope sequence inserted between 3757 and 3758 was ordered from Integrated DNA Technologies (Coralville, Iowa). Plasmid pAAV RC2/6-Q585Myc was created by inserting the gblock fragment into pAAV RC2/6 digested with MscI (New England Biolabs, cat. no. R0534L) and AflII (New England Biolabs, cat. no. R0520L) by ligation-independent cloning as described in (2012) Methods Mol. Biol. 52:51–9.

To create pAAV RC9-A589Myc, a first polymerase chain reaction (PCR) product comprising (5' to 3') a BsiW1 restriction site, the nucleotide sequence between positions 3052 and 3779 of pAAV RC9, and a nucleotide sequence overhanging the c-myc epitope, and a second PCR product comprising (5' to 3') a nucleotide sequence overhanging the c-myc epitope, the nucleotide sequence between positions 3779 to 4404 of pAAV RC9, and a Pme1 restriction site were created using the primers set forth in Table 2.

Plasmid pAAV RC9-A589Myc (i.e., a pAAV RC2/9 plasmid modified to encode the c-myc epitope between amino acids A589 and Q590 of the VP1 capsid protein) was created by digesting pAAV RC9 with BsiW1 (New England Biolabs, R0553L) and Pme1 (New England Biolabs, R0560L), and inserting the two PCR products by ligation-independent cloning as described in (2012) Methods Mol. Biol. 52:51–9.

Table 2

pscAAV-CMV-eGFP was produced by introducing the GFP fragment into the pscAAV MCS vector (Cell Biolabs, cat. no. VPK-430) using the BamHI and NotI restriction site. Plasmid pAAV-EF1a-eGFP and pAAV-CAGG-eGFP were made by de novo synthesis by Thermofisher Scientific (Waltham, MA).

Example 2: Antibody-mediated retargeting of AAVin Vitro viral vectors

HepG2 is a human breast cancer cell line, which expresses the liver-specific marker asialoglycoprotein receptor 1 (ASGR1). To test whether scAAV2-N587Myc-CMV-hrGFP viral vectors can infect HepG2 cells, for example, via a bispecific antibody that recognizes both c-myc and ASGR1 (anti-myc-ASGR1 antibody), mixtures comprising different ratios of the number of viral genomes (5e9 viral genomes) to the number of antibody molecules (1:0.5, 1:1, 1:2, 1:4, 1:8, 1:15, 1:20, 1:50, or 1:100) of scAAV2-N587Myc-CMV-eGFP and the anti-myc-ASGR1 bispecific antibody were incubated at room temperature for half an hour and then added to HepG2 cells. HepG2 cells were also incubated with wild-type scAAV2-CMV-eGFP viral vectors alone, scAAV2-N587Myc-CMV-eGFP viral vectors alone, or scAAV2-N587Myc-CMV-eGFP viral vectors with monospecific anti-myc antibody (Regeneron Pharmaceuticals, Inc., Tarrytown, NY) at a 1:8 ratio as controls. Three days postinfection, GFP expression by infected HepG2 cells was confirmed by fluorescence-activated cell sorting (FACS) (Figure 1). GFP expression was also detected in a comparable percentage of HepG2 cells when incubated with wild-type scAAV2-CMV-eGFP and when incubated with scAAV2-N587Myc-CMV-eGFP viral vectors and anti-myc-ASGR1 bispecific antibody at a 1:8 ratio (44.6% and 44.1%, respectively, Figures 1A and 1G, top and bottom panels). Lower GFP expression was detected by FACs in HepG2 cells incubated with a mixture of scAAV2-N587Myc-CMV-eGFp viral vectors and either top or bottom bispecific anti-myc-ASGR1 antibody (Regeneron Pharmaceuticals, Inc., Tarrytown, NY) (Figure 1C, bottom panel). Additionally, GFP was not detected in HepG2 cells infected with scAAV-N587Myc-CMV-eGFP in the absence of anti-myc-ASGR1 antibody, nor in HepG2 cells incubated with scAAV2-N587Myc-CMV-eGFP and monospecific anti-myc antibody.

Similarly, 293T-hASGR1 cells, which are 29T3 cells genetically modified to express human (h)ASGR1 on the cell surface, were incubated with mixtures comprising different ratios of viral genomes to the number of antibody molecules (1:0.5, 1:1, 1:2, 1:4, 1:8, 1:15, 1:20, or 1:100) of scAAV2-N587Myc-CMV-eGFP and bispecific anti-myc-ASGR1 antibody. Wild-type 293T cells (not expressing hASGR1) incubated with a mixture of scAAV2-N587Myc-CMV-eGFP and anti-myc-ASGR1 bispecific antibody at a 1:8 ratio, and 293T-hASGR1 cells incubated with (a) wild-type scAAV viral vectors only, (b) scAAV2-N587Myc-CMV-eGFP viral vectors only, or (c) scAAV2-N587Myc-CMV-eGFP viral vectors with an irrelevant bispecific anti-myc-GCGR antibody that recognizes c-myc and glucagon receptor (GCGR) (Regeneron Pharmaceuticals, Inc., Tarrytown, NY) not expressed by 293T cells at a 1:8 ratio, served as controls. Three days postinfection, 293T-hASGR1 or 293T cells were stained with anti-human hASGR1 antibody (Regeneron Pharmaceuticals, Inc., Tarrytown, NY) followed by APC-conjugated goat anti-human antibody (Jackson ImmunoResearch Laboratory Inc, cat. no. 109-136-098, West Grove, PA) and GFP expression analyzed by FACS. hASGR1 expression was detected on the surface of 293T-hASGR1 cells (Figures 2Ai-2Ax and 2Axii) but not 293T cells (Figure 2Axi). GFP-positive 293T-hASGR1 cells were detected after incubation with scAAV2-N587Myc-CMV-eGFP and anti-myc-ASGR1 bispecific antibody mixtures at ratios of 1:0.5, 1:1, 1:2, 1:4, 1:8, 1:15, 1:20, and 1:50 at levels (56.9% to 68.3%) comparable to 293T-hAsGR1 cells incubated with wild-type scAAV (56.7%) (Figures 2Ai and 2Aiii–2Aix). A 1:100 ratio of scAAV2-N587Myc-CMV-eGFP and anti-myc-ASGR1 bispecific antibody decreased infectivity (Figure 2Ax). Incubation with scAAV2-N587Myc-CMV-eGFP alone or scAAV-N587Myc with an irrelevant bispecific anti-myc-GCGR antibody did not result in GFP expression by 293T-hASGR1 cells (Figures 2Aii and 2Axii).

In a similar experiment, 293T-hASGR1 cells were incubated with cells incubated with unmodified AAV9-CAGG-GFP viral vectors alone, AAV9-A589Myc-CAGG-eGFP viral vectors alone, or AAV9-A589Myc-CAGG-eGFP viral vectors mixed with anti-Myc-ASGR1 bispecific antibodies at different ratios. Three days postinfection, GFP expression was analyzed by FACS. GFP-positive 293T-hASGR1 cells were detected after incubation with AAV9-A589Myc-CAGG-eGFP and anti-myc-ASGR1 bispecific antibody mixtures at ratios of 1:1, 1:2, 1:4, 1:8, 1:20, 1:50, and 1:100 at levels (41.1% to 91%) comparable to 293T-hASGR1 cells incubated with wild-type AAV9-CAGG-eGFP (9.72%) (z and Figures 2B(iii)–(ix)). Incubation with AAV9-A589Myc-CAGG-eGFP alone did not result in GFP expression by 293T-hASGR1 cells (Figure 2B(ii)).

The ability of a bivalent anti-hASGR1 antibody (Regeneron Pharmaceuticals, Inc., Tarrytown, NY) to block infection of 293T-hASGR1 cells was tested. 293T-hASGR1 cells were incubated with the bivalent anti-hASGR1 antibody at different concentrations for 1 h at room temperature and subsequently incubated with a mixture comprising a 1:8 ratio of scAAV2-N587Myc-CMV-eGFP and bispecific anti-myc-ASGR1 antibody. Three days post-infection, cells were fixed and analyzed by FACS as described above. Figure 3 provides data showing that scAAV2-N587Myc-CMV-eGFP entry can be blocked in a dose-dependent manner by a bivalent anti-hASGR1 antibody.

To determine whether sequential administration of bispecific antibodies and modified AAV could result in antibody-mediated retargeting of the modified AAV, different numbers of bispecific anti-myc-ASGR1 antibody molecules (ranging from 1x109 to 1x1012 molecules) were added to 2x105 293T-hASGR1 cells for one hour, after which 1x109 scAAV-N587Myc viral vectors were added. Controls included (1) 293T-hASGR1 cells incubated with wild-type scAAV only, i.e., in the absence of any compound, (2) 293T-hASGR1 cells sequentially incubated with 1x1011 irrelevant bispecific anti-myc-GCGR antibody molecules and 1x109 scAAV2-N587Myc-CMV-eGFP viral vectors, and (3) 293T cells, which do not express hASGR1, sequentially incubated with 1x1011 bispecific anti-myc-ASGR1 antibody molecules and 1x109 scAAV-N587Myc viral vectors. Two days post-infection, GFP expression by infected 293T-hASGR1 cells was visualized by microscopy (Figure 4). GFP expression by 293T-hASGR1 cells was observed after incubation with wild-type scAAV alone and after sequential incubation with anti-myc-ASGR1 antibody molecules at all concentrations and scAAV2-N587Myc-CMV-eGFP (Figures 4A, 4C-4I). No GFP was detected in 29T3-hASGR1 cells after incubation with scAAV2-N587Myc-CMV-eGFP alone (Figure 4B), in 293T cells not expressing ASGR1 after sequential incubation with anti-myc-hASGR1 antibody and scAAV2-N587Myc-CMV-eGFP viral vectors (Figure 4J), or in 29T3-hASGR1 cells after sequential incubation with irrelevant bispecific anti-myc-GCGR antibody and vectors. scAAV2-N587Myc-CMV-eGFP viral (Figure 4K).

As described herein, self-complementary AAV (scAAV) tropism can be (1) inactivated, e.g., by modification of a capsid protein, e.g., by insertion of a c-myc epitope, and optionally, (2) redirected using bispecific antibodies, e.g., with a bispecific antibody that recognizes the c-myc epitope and a ligand expressed by the target cell. To determine whether single-stranded AAV (ssAAV) tropism can similarly be (1) reduced or abrogated and optionally (2) redirected, 293T-hASGR1 cells were incubated with ssAAV2-N587Myc-CMV-hrGFP viral vectors produced as described in Example 1 in the absence or presence of bispecific anti-myc-hASGR1 antibody at different viral vector:antibody ratios (1:0, 1:1, 1:2, 1:4, 1:8, 1:20, 1:100, or 1:1000). Controls included 293T-hASGR1 cells incubated with wild-type ssAAV, 293T-hASGR1 cells incubated with ssAAV2-N587Myc-CMV-hrGFP viral vectors and irrelevant bispecific anti-myc-GCGR antibody at a viral vector:antibody ratio of 1:8, and 293T cells (not expressing hASGR1) incubated with ssAAV2-N587Myc-CMV-hrGFP viral vectors and bispecific anti-myc-hASGR1 antibody at a viral vector:antibody ratio of 1:8. Three days postinfection, cells were fixed and GFP expression was detected by FACS (Figure 5). GFP expression was detected in 293T-hASGR1 cells incubated with wild-type ssAAV and with mixtures of ssAAV2-N587Myc-CMV-hrGFP viral vectors and anti-myc-ASGR1 bispecific antibodies at all ratios (Figures 5A, 5C-5I). GFP expression was not observed in 29T3 cells incubated with ssAAV2-N587Myc-CMV-hrGFP viral vectors and anti-myc-ASGR1 bispecific antibodies (Figure 5J), or by 293T-hASGR1 cells incubated with ssAAV2-N587Myc-CMV-hrGFP viral vectors alone, or with ssAAV-N587Myc viral vectors and irrelevant bispecific anti-myc-GCGR antibody (Figures 5B, 5K).

The human (h) glucagon receptor (GCGR) is not normally expressed in 293T cells. However, 293T-hGCGR is a stable 293T cell line genetically modified to express hGCGR on the cell surface. To test whether retargeting of scAAV2-N587Myc-CMV-eGFP viral vectors can be mediated by a bispecific anti-myc-GCGR antibody through the hGCGR receptor, 293T-hGCGR cells were incubated with different mixtures comprising different ratios of scAAV2-N587Myc-CMV-eGFP:anti-myc-GCGR bispecific antibody. scAAV2-N587Myc-CMV-eGFP viral vectors were mixed with bispecific antimyc-GCGR antibody at ratios of 1:0.5, 1:1, 1:2, 1:4, 1:8, 1:15, 1:20, 1:50, or 1:100 at room temperature for half an hour before addition to 293T-hGCGR cells. Three days postinfection, GFP expression by infected 293T-hCGCR cells was confirmed by fluorescence-activated cell sorting (FACS) (Figure 6). A significant percentage of GFP-positive cells were detected after incubation with wild-type scAAV (Figure 6A) or scAAV2-N587Myc-CMV-eGFP/anti-myc-GCGR bispecific antibody at a ratio of 1:0.5, 1:1, 1:2, 1:4, 1:8, 1:15, 1:20, 1:50, and 1:100 (Figures 6C–6K). No GFP was detected in 293T-hGCGR cells incubated with scAAV2-N587myc-CMV-eGFP viral vectors alone or scAAV2-N587Myc-CMV-eGFP viral vectors with anti-myc monospecific antibody (Figures 6B and 6L, respectively).

Jurkat is a human acute T-cell leukemia cell line, expressing human (h) CD3. To test whether the targeting of AAV6-Q585Myc-EF1a-eGFP viral vectors can be mediated by bispecific anti-myc-CD3 antibody through the hCD3 receptor, Jurkat cells were incubated with different mixtures comprising different ratios of AAV6-Q585Myc-EF1a-eGFP:bispecific anti-myc-CD3 antibody. AAV6-Q585Myc-EF1a-eGFP viral vectors were mixed with bispecific anti-myc-CD3 antibody at ratios of 1:1, 1:5, 1:10, 1:100, 1:1000 at room temperature for half an hour before the addition of Jurkat cells. Three days post-infection, GFP expression by infected Jurkat cells was confirmed by fluorescence-activated cell sorting (FACS) (Figure 7). A significant percentage of GFP-positive cells were detected after incubation with wild-type AAV6-EF1a-eGFP (Figure 7B) or AAV6-Q585Myc-EFlaeGFP/anti-myc-CD3 bispecific antibody at a ratio of 1:1, 1:5, 1:10, and 1:100 (Figures 7D–7G). No GFP was detected in Jurkat cells incubated with AAV6-Q585Myc-EF1a-eGFP viral vectors (Figures 7C).

This example describes the inactivation of the natural tropism of several self-complementary (sc) or single-stranded (ss) AAV serotypes as demonstrated by the inability of genetically engineered scAAV2 or ssAAV2 with a c-myc epitope inserted between amino acids N587 and R588 of the VP1 capsid protein (scAAV-N587myc or ssAAV-N587myc), AAV6 with a c-myc epitope inserted between Q585 and S586, and AAV9 to infect cells normally infected by wild-type AAV (Figures 1A-1B, 2A-2B, 3A-3B, 4A-4B, 5A-5B, 6A-6B, and 7). Furthermore, this example demonstrates the ability of a bispecific antibody that recognizes the c-myc epitope and a second ligand expressed by the target cell (e.g., hASGR1, hGCGR, or hCD3) to redirect the tropism of the modified AAV and mediate infection of the target cell by pseudotyped AAV in a ligand-specific manner (Figures 1-7). Furthermore, this example demonstrates that simultaneous administration of the genetically modified sc- or ss-AAV and the bispecific antibody is not necessary for infection, i.e., sequential administration is sufficient for redirection of the genetically modified sc- or ss-AAV in vitro (Figure 4).

Example 3: Antibody-mediated retargeting of modified viral vectors in vivo

Redirection of viral vectors to the liver with different formats of multispecific binding molecules

To determine whether anti-myc-ASGR1 bispecific antibody could redirect scAAV2-N587myc-CMV-eGFP viral vectors to hASGR1-expressing liver cells in vivo, mice genetically modified to express hASGR1 in their liver cells on the C57BL/6 background and control wild-type C57BL/6 mice were injected with 1x1011 (titrated by qPCR) of wild-type scAAV2-CMV-eGFP viral vectors alone or scAAV2-N587myc-CMV-eGFP together with anti-myc-ASGR1 bispecific antibody at a 1:8 ratio of viral genome to antibody molecules intravascularly. Controls included mice injected with saline [250 mM NaCl] or with scAAV2-N587myc-CMV-eGFP viral vector alone. Ten days after injection, mice were sacrificed and transcardially perfused with 4% PFA. Organs including the liver, kidney, and heart were removed and dehydrated in 15% sucrose followed by 30% sucrose. Organs were then cryosectioned onto slides and stained with chicken anti-EGFP antibody (Jackson ImmunoResearch Labs, Inc. West Grove, PA) and Alexa-488-conjugated anti-chicken secondary antibody (Jackson ImmunoResearch Labs, Inc. West Grove, PA). (Figures 8A–8C). GFP-positive cells were detected in livers of those transgenic animals modified to express ASGR1 in the liver and injected with wild-type scAAV2-CMV-eGFP or scAAV2-N587myc-CMV-eGFP together with an anti-myc-ASGR1 bispecific antibody (Figures 8A(i) and 8A(iv)), and in livers of wild-type C57BL/6 mice injected with wild-type scAAV2-CMV-eGFP (Figure 8A(v)). No GFP was detected in any spleen or kidney samples (Figures 8B and 8C), nor in liver, spleen, or kidney samples from any animal injected with saline or scAAV2-N587myc-CMV-eGFP viral vectors alone (Figures 8A (ii, iii, vi, vii)), nor in liver samples taken from wild-type C57BL/6 animals injected with scAAV2-N587myc-CMV-eGFP together with anti-myc-ASGR1 bispecific antibody (Figure 8A (viii)). In summary, the combination of scAAV2-N587myc-CMV-eGFP viral vectors and bispecific anti-myc-ASGR1 antibody infected only those (liver) cells expressing hASGR1, strongly suggesting that the scAAV2-CMV-eGFP viral vector was inactivated by the capsid protein modification, i.e. the natural tropism of the scAAV viral vector could be reduced or abrogated, e.g. with a c-myc epitope, and such viral vectors could be specifically reactivated, e.g. specifically redirected, e.g. to liver cells, in vivo, by anti-myc-ASGR1 bispecific antibodies.

Similarly, to determine whether anti-myc-ASGR1 bispecific antibody could redirect ssAAV2-N587myc-CAGG-eGFP viral vectors to hASGR1-expressing liver cells in vivo, mice genetically modified such that their liver cells express hASGR1 on the C57BL/6 background and control wild-type C57BL/6 mice were injected with 2.18x1011 (titrated by qPCR) of wild-type ssAAV2-CAGG-eGFP viral vectors alone or ssAAV2-N587myc-CAGG-eGFP together with anti-myc-ASGR1 bispecific antibody and at a 1:4 ratio of viral genome to antibody molecules intravascularly. Controls included mice injected with PBS or ssAAV2-N587myc-CAGG-eGFP viral vector alone. Four weeks after injection, mice were euthanized and transcardially perfused with 4% PFA. Organs including the liver, kidney, and heart were removed and dehydrated in 15% sucrose followed by 30% sucrose. Organs were then cryosectioned onto slides and stained with chicken anti-EGFP antibody (Jackson ImmunoResearch Labs, Inc. West Grove, PA) and Alexa-488-conjugated anti-chicken secondary antibody (Jackson ImmunoResearch Labs, Inc. West Grove, PA). GFP-positive cells were detected in livers of transgenic animals engineered to express ASGR1 in the liver and injected with wild-type ssAAV2-CAGG-eGFP or ssAAV2-N587myc-CAGG-eGFP together with an anti-myc-ASGR1 bispecific antibody (Figures 9E-9F, 9P-9R), and in livers of wild-type C57BL/6 mice injected with wild-type ssAAV2-CAGG-eGFP (Figure 9B-9C). Strikingly, the infection efficiency of ssAAV2-N587myc-CAGG-eGFP together with the bispecific anti-myc-ASGR1 antibody is much higher than that of WT ssAAV2-CAGG-GFP (Figures 9E-9F, 9P-9R). GFP was not detected or was barely detected in liver samples from any animal injected with saline or ssAAV2-N587myc-CAGG-eGFP viral vectors alone (Figures 9A, 9D, 9G-9L), nor in liver samples taken from wild-type C57BL/6 animals injected with ssAAV2-N587myc-CAGG-eGFP together with anti-myc-ASGR1 bispecific antibody (Figure 9M-9O). In summary, the combination of ssAAV2-N587myc-CAGG-eGFP viral vectors and bispecific anti-myc-ASGR1 antibody infected only those (liver) cells expressing hASGR1, strongly suggesting that the ssAAV2-N587myc-CAGG-eGFP viral vector was inactivated by the capsid protein modification, i.e. the natural tropism of the scAAV viral vector could be reduced or abrogated, e.g. with a c-myc epitope, and such viral vectors could be specifically reactivated, e.g. specifically redirected, e.g. to liver cells, in vivo, by anti-myc-ASGR1 bispecific antibodies.

Additional experiments were performed using a pseudotyped AAV9 viral vector using mice genetically modified such that their liver cells expressed hASGR1 on the C57BL/6 background. These mice were injected with 1x1011 (titrated by qPCR) of wild-type AAV9-CAGG-eGFP or AAV9-A589myc-CAGG-eGFP viral particles together with anti-myc-ASGRI bispecific antibody at a 1:100 ratio of viral genome to antibody molecules intravascularly. Controls included mice injected with PBS or with AAV9-N587myc-CAGG-eGFP viral particles together with an irrelevant bispecific anti-myc-hCD3 antibody. Four weeks after injection, mice were sacrificed. Livers were fixed with 10% formalin and shipped to HistoWiz. Inc (New York, NY) for GFP staining. GFP-positive cells were detected in livers of animals injected with wild-type AAV9-CAGG-eGFP or AAV9-N587myc-CAGG-eGFP viral particles together with an anti-myc-ASGR1 bispecific antibody (Figures 10A and 10D). GFP was either not detected or was barely detected in liver samples from any animal injected with saline or with AAV9-N587myc-CAGG-eGFP viral particles together with an anti-myc-hCD3 bispecific antibody, respectively (Figures 10B and 10C). In summary, similar to AAV2, the natural tropism of AAV9 can be reduced or abrogated by modifying the capsid protein, e.g., by inserting a c-myc epitope, and such a modified viral vector, e.g., AAV9-A589myc, can be redirected to specific cell types using the corresponding bispecific anti-epitope cell-specific marker binding protein, e.g., bispecific anti-myc-ASGR1 antibody against liver cells.

Anti-myc scFc added to the “button-in-button” Fc format of anti-hASGR1 IgG4

To determine whether various formats of multispecific binding molecules could mediate AAV infection, anti-myc scFv was fused to the C-terminus of a knob-shaped anti-hASGR1 IgG4 antibody chain (see Figure 11A). Specifically, Integrated DNA technologies (Skokie, Illinois) provided a gblock encoding an anti-myc scFv (SEQ ID NO:28) flanked by arms homologous to 1459-1478 and 1479-1498 of pRG7078 encoding an anti-hASGR1 knob-shaped IgG4 heavy chain. The nucleotide (NA) sequences encoding and amino acid (AA) sequences of HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the scFv (SEQ ID NO:37) encoded by<s>E<q>ID NO:28 are provided in Table 3.

Table 3: SEQ ID NOs associated with the scFv established as SEQ ID NO:37

The gblock was cloned into pRG7078 encoding an anti-hASGR1 hidden-knob IgG4 heavy chain to produce pRG7078 encoding an anti-hASGR1 hidden-knob IgG4 scFv fusion polypeptide “anti-hASGR1-IgG4-Fc/anti-myc bispecific”. The c-myc modified plasmid pRG7078, the unmodified anti-hASGR1 IgG4 hidden-knob pRG7078, and a plasmid containing an antibody light chain were transfected into 293T cells cultured with OPTI-MEM (cat. no. 31985070, Thermo Fisher). Four days after transfection, the medium was collected and the anti-hASGR1-IgG4-Fc/anti-myc bispecific binding molecule was purified using a protein A column (cat. no. 89948, Thermo Fisher).

To create a pseudotyped AAV8 viral vector, pAAV-RC8 N590myc, a gblock with a c-myc epitope (SEQ ID NO:22) inserted between N590 and T591 of an AAV8 VP1 capsid protein was ordered from Integrated DNA technologies (Skokie, Illinois). Plasmid pAAV RC8 (SEQ ID NO:23) was digested with Mlu1 and Sbf1 enzymes and the gblock was cloned into the vector using SLIC to yield pAAV RC8 N590myc (SEQ ID NO:24).

293T-hASGR1 cells, which are 293T cells genetically modified to express human (h)ASGR1 on the cell surface, were incubated with mixtures comprising different ratios of AAV8-N590Myc-CAGG-eGFP to anti-hASGR1-IgG4-Fc/anti-myc bispecific binding molecule molecules (1:0, 1:1, 1:2, 1:4, 1:8, 1:12, 1:15, 1:50, or 1:100) or AAV8-CAGG-eGFP viral genomes. Three days postinfection, GFP expression was analyzed by FACS. GFP-positive 293T-hASGR1 cells were detected after incubation with mixtures of AAV8-N590Myc-CAGG-eGFP and anti-hASGRI-IgG4-Fc/anti-myc bispecific molecules at ratios of 1:1, 1:2, 1:4, 1:8, 1:12, 1:15, 1:50, or 1:100 (2.12% to 22.2%; Figures 12D–12K) comparable to 293T-hASGR1 cells incubated with wild-type AAV8-CAGG-eGFP (46.1%) (Figure 12A). Neither mock-infected cells (Figure 12B) nor incubation with AAV8-N590Myc-CAGG-eGFP alone resulted in GFP expression by 293T hASGR1 cells (Figure 12C).

To determine whether anti-hASGR1-IgG4-Fc/anti-myc bispecific binding proteins could redirect AAV8 N590myc-CAGG-eGFP viral particles to hASGR1-expressing liver cells in vivo, mice genetically modified such that their liver cells express hASGR1 on the C57BL/6 background were injected intravascularly with 1x1011 (titrated by qPCR) of wild-type AAV8-CAGG-eGFP or AAV8 N590myc-CAGG-eGFP viral particles together with anti-hASGR1-IgG4-Fc/anti-myc bispecific binding molecules at a 1:12 ratio of viral genome to binding protein molecules. Control animals were injected with AAV8 N590myc-CAGG-eGFP viral particles together with an irrelevant anti-hCD3xanti-myc bispecific binding molecule that had a similar format to the anti-hASGR1-IgG4-Fc/anti-myc bispecific binding molecule. Three weeks after injection, mice were sacrificed and transcardially perfused with 4% PFA. Organs such as the liver, kidney, and heart were removed and dehydrated in 15% sucrose followed by 30% sucrose. Organs were then cryosectioned onto slides and stained with chicken anti-EGFP antibody (Jackson ImmunoResearch Labs, Inc. West Grove, PA) and Alexa 488-conjugated anti-chicken secondary antibody (Jackson ImmunoResearch Labs, Inc. West Grove, PA). GFP-positive cells were detected in livers from animals injected with wild-type AAV8-CAGG-eGFP (Figures 13A-13C) or AAV8 N590myc-CAGG-eGFP viral particles together with the bispecific anti-hASGRI-IgG4-Fc/anti-myc binding molecules (Figures 13G-13I). GFP was either not detected or barely detected in liver samples from any animal injected with AAV8 N590myc-CAGG-eGFP viral particles together with the irrelevant anti-hCD3/myc binding protein (Figures 13D-13F). In summary, epitope-tagged AAV8, e.g., AAV8-N590myc, can be redirected to a specific cell type using a bispecific binding molecule that specifically binds to the epitope and a cell-specific marker expressed by the targeted cell type.

Targeting of viral vectors to the intestine and/or pancreas

293T cells engineered to express human ectonucleoside triphosphate diphosphohydrolase 3 (hENTPD3) on the cell surface ("293T-ENTPD3") were incubated with mixtures comprising different ratios of AAV2-N587Myc-CAGG-eGFP or AAV2-CAGG-GFP viral genomes and bispecific binding molecule formed by fusing anti-myc scFv to the C-terminus of one chain of a "buttonhole" anti-hENTPD3 IgG4 antibody (anti-hENTPD3-IgG4-Fc/anti-myc) in different ratios (1:0, 1:1, 1:2, 1:4, 1:8, 1:20, 1:50, 1:100, or 1:200). Three days postinfection, g Fp expression was analyzed by FACS. GFP-positive 293T-ENTPD3 cells were detected after incubation with AAV2-N587Myc-CAGG-eGFP and anti-hENTPD3-IgG4-Fc/anti-myc mixtures at levels (1.48% to 16%; Figures 14C–14J) comparable to 293T-ENTPD3 cells incubated with wild-type AAV2-CAGG-eGFP (66%) (Figure 14A). Incubation with AAV2-N587Myc-CAGG-eGFP alone results in a very low level of GFP expression by 293T-ENTPD3 cells (Figure 14B).

ENTPD3 was determined to be expressed in the intestinal mucosa (data not shown). To determine whether anti-hENTPD3-IgG4-Fc/anti-myc binding protein could redirect AAV2-N587myc-CAGG-eGFP viral particles to ENTPD3-expressing intestinal cells in vivo, C57BL/6 mice were intravascularly injected with 5x1011 (titered by qPCR) AAV2-N587myc-CAGG-eGFP viral particles together with anti-hENTPD3-IgG4-Fc/anti-myc binding proteins at a 1:20 ratio of viral genome to binding protein molecules. Control mice were injected with PBS, wild-type AAV9, or with AAV2-N587myc-CAGG-eGFP viral particles together with an irrelevant binding protein molecule. Three weeks after injection, mice were sacrificed. Livers, intestines, and pancreases were fixed with 10% formalin and sent to HistoWiz. Inc. (New York, NY) for GFP staining. GFP-positive cells were detected in mice injected with wild-type AAV9 (Figure 15B(ii)), but not in livers of mice injected with PBS or AAV2-N587myc-CAGG-eGFP viral particles together with irrelevant binding molecules or anti-hENTPD3-IgG4-Fc/anti-myc binding proteins (Figure 15A(iii)–15A(iv), respectively), indicating that AAV2-N587myc viral vectors do not infect ENTPD3-negative liver cells even when co-injected with hENTPD3-IgG4-Fc/anti-myc binding proteins. GFP was detected in intestines only of mice injected with wild-type AAV9 or AAV2-N587myc-CAGG-eGFP viral particles together with hENTPD3-IgG4-Fc/anti-myc binding proteins (Figures 15B(i)-15B(iv)). GFP is detected in the pancreatic islets of a mouse injected with AAV2-N587myc-CAGG-eGFP viral particles together with anti-hENTPD3-IgG4-Fc/anti-myc binding proteins (Figure 15C(iv)). WT AAV9 infected both islet and non-islet cells of the pancreas (Figure 15C(ii)). No GFP was detected in pancreas samples from mice injected with saline or AAV2-N587myc-CAGG-eGFP viral particles together with irrelevant binding proteins (Figures 7C(i) and 7C(iii)). In summary, the natural tropism of AAV can be reduced or abrogated by inserting a heterologous epitope and retargeting to the same or other cells using a multispecific binding protein that binds to the epitope tag and a marker expressed by the retargeted cells or tissues.

This example demonstrates that several different serotypes of adeno-associated viral vector can be genetically engineered with a heterologous epitope (e.g., c-myc) as described herein to inactivate infectivity, and a bispecific antibody (regardless of bispecific formats) that recognizes both the adeno-associated viral vector and the heterologous epitope (e.g., c-myc) and a marker expressed by a target cell can be used to redirect the viral vector to deliver a nucleotide of interest.

Example 4: Use of genetically modified AAV-N587Myc viral vectors to deliver therapeutic cargo to specific cells

This example demonstrates the ability of AAV-N587Myc viral vectors to specifically deliver therapeutic cargo, such as one or more suicide genes, a biological therapeutic (e.g., antibody), a CRISPR/Cas gene editing system, shRNA, etc., to a target cell type. Specifically, this example describes the delivery of a suicide gene, antibody-encoding sequence, or CRISPR/Cas gene editing system to cells expressing a target ligand.

Delivery of a suicide gene to cells expressing a target ligand

To test the ability of scAAV2-N587Myc viral vectors to deliver a suicide gene to a specific cell, a HER2+ breast cancer xenograft nude mouse model described by Wang et al. ((2010) Cancer Gene Therapy 17:559-570) was used.

Viral vectors: scAAV2-N587Myc or ssAAV2-N587Myc viral vectors carrying an EGFP reporter gene (scAAV2-N587Myc-EGFP or ssAAV2-N587MycEGFP) are generated as described in Example 1. scAAV2-N587Myc or ssAAV2-N587Myc viral vectors carrying a suicide gene (SG) are generated similarly. Briefly, 293T17 cells are transfected with (1) pAd helper and (2) pAAV RC2 (encoding a wild-type capsid) or pAAV RC2-N587Myc (encoding a modified capsid with a c-myc epitope) vectors as described above, and with (3) a pAAV vector carrying a suicide gene, e.g., cytosine deaminase gene, the herpes simplex virus thymidine kinase gene, under the control of a promoter, e.g., CMV. ssAAV-N587MycSG or scAAV-N587SG viral vectors are isolated and titered as described in Example 1.

Cell lines: BT474 breast cancer cell line, SK-BR-3 breast cancer cell line, and Calu-3 lung cancer cell line are HER2-positive human tumor cell lines (Bunn P.A.et al.,(2001)Clin. Cancer Res.

7:3239-3250; Pegram M,et al.(1999)Oncogene18:2241-2251; Spiridon CI,et al.,(2002)Clin. Cancer Res.8:1720-1730). A-673 rhabdomyosarcoma and HeLa cervical cancer are HER2-negative human tumor cell lines and BEAS-2b is an immortalized HER2-negative bronchial epithelial cell line (Jia LTet al.(2003)Cancer Res.63:3257-3262; Kern JA,et al.,(1993)Am. J. Respir. Cell Mol. Biol.9:448-454; Martinez-Ramirez A,et al.,(2003) Cancer Genet. Cytogenet. 2003; 141:138-142). All these cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in the medium recommended by the ATCC.

Mice: Female nude mice, 6- to 8-weeks old, were obtained and housed under specific pathogen-free conditions. On day 0, mice were simultaneously injected (1) subcutaneously in the right flank with 107 BT474, SK-BR-3, Calu-3, A-673, or HeLa tumor cells and (2) treated intravenously with anti-myc-HER2 bispecific antibody and ss- or scAAV-N587Myc viral vectors carrying a reporter gene (e.g., EGFP) or suicide gene. Controls include untreated animals (animals injected with tumor cells only), animals injected with wild-type ss- or scAAV viral vectors carrying a reporter or suicide gene, animals injected with anti-myc-HER2 bispecific antibody only, or animals injected with ss- or scAAV-N587Myc viral vectors carrying a reporter or suicide gene only. All animals are treated with an appropriate prodrug 1 day after injection and treatment. The size of each tumor is measured with calipers twice weekly and tumor volume is calculated as lengthxwidth2x0.52. Following morbidity, tumor ulceration, a tumor diameter of 15 mm, or a tumor volume of 1000 mm3 , mice are sacrificed and the date of sacrifice is recorded as the date of death. Livers, spleens, kidneys, and tumors from animals injected with wild-type ss- or scAAV or ss- or scAAV-N587Myc virus vectors carrying a reporter gene are fixed and reporter gene expression is visualized.

Although targeted delivery of suicide-inducing genes has been described (Zarogoulidis P.,et al.(2013)J. Genet. Sindr. Gene Ther.4:16849), this example describes delivery of a suicide gene to a cell expressing the target HER2 ligand using the viral vectors described herein comprising a heterologous epitope, e.g., c-myc, and a bispecific antibody that specifically binds both the target ligand and the heterologous epitope. In additional experiments, a suicide gene is delivered to another cell type expressing one or more different target ligands using a viral vector comprising a heterologous epitope as described herein and a bispecific antibody that specifically binds both the heterologous epitope (e.g., c-myc) and the target receptor. Illustrative and non-limiting examples of suitable receptors to target include those receptors that mediate endocytosis of the viral vector, for example, carcinoembryonic antigen (CEA) (Qiu Y,et al.(2012)Cancer Lett.316:31-38) and vascular endothelial growth factor receptor (VEGf R) (Leng A,et al.(2013)Tumour Biol.32:1103-1111; Liu T,et al.(2011)Exp. Mol. Pathol.91:745-752). Additional receptors that can be targeted include: epidermal growth factor receptor (EGFR) (Heimberger AB,et al.(2009)Expert Opin. Biol. Ther.9:1087-1098), cluster of differentiation 44s (CD44s) (Heider KH,et al.(2004)Cancer Immunol. Immunother.53:567-579), cluster of differentiation 133 (CD133 also known as AC133) (Zhang SS,et al.(2012)BMC Med.;10:85), folate receptor (FR) (Duarte S,et al.,(2011)J. Control Release149(3):264-72), transferrin receptor (TfR), or cluster of differentiation 71 (CD71) (Habashy HO,et al., Breast Cancer Res. Treat.119(2):283-93), mucins (Torres MP,et al.,(2012)Curr. Pharm. Des.2012; 18(17):2472-81), phase-specific embryonic antigen 4 (SSEA-4) (Malecki M.,et al.,(2012)J. Stem Cell Res. Ther.2(5), tumor resistance antigen 1-60 -1-60) (Malecki M.,et al.,(2013)J. Stem Cell Res. Ther.3:134).

Claims (21)

1. A composition comprising (i) a recombinant adeno-associated virus (AAV) vector comprising an AAV capsid encapsulating a nucleotide of interest, wherein the AAV capsid comprises a recombinant AAV capsid protein modified to comprise a heterologous epitope, wherein the recombinant AAV capsid protein is derived from an AAV capsid gene modified to express the heterologous epitope; (ii) a multispecific binding molecule comprising (a) an antibody paratope that specifically binds to the heterologous epitope; and (b) a retargeting ligand that specifically binds to a protein or cell surface marker; and optionally (iii) a pharmaceutically acceptable carrier, where the AAV capsid presents: - tropism reduced to null in the absence of the multispecific binding molecule; and - modified tropism, upon combination with the multispecific binding molecule, compared to a reference AAV capsid, wherein the reference AAV capsid comprises a reference AAV capsid protein that is identical to the recombinant AAV capsid protein except for the lack of the heterologous epitope. 2. The composition of claim 1, wherein the recombinant AAV capsid protein further comprises a mutation in addition to the heterologous epitope. 3. The composition of claim 1 or 2, wherein the heterologous epitope is inserted such that the insertion partially reduces the tropism of the AAV capsid compared to a reference AAV capsid lacking the heterologous epitope and the AAV capsid protein further comprises a mutation (e.g., a substitution, deletion, insertion other than the insertion of the heterologous epitope), in addition to the insertion of the heterologous epitope, the mutation further reducing and/or abolishing the tropism of the recombinant AAV capsid compared to a reference AAV capsid lacking the mutation. 4. The composition of any one of claims 1 to 3, wherein the modified tropism upon combination with the multispecific binding molecule means that the AAV capsid tropism is restored and redirected compared to the reference AAV capsid tropism. 5. The composition of any one of the preceding claims, wherein (i) the heterologous epitope comprises an affinity tag and an optional linker, and/or (ii) the recombinant AAV capsid protein comprises an amino acid sequence EQKLISEEDL (set forth as SEQ ID NO: 6) flanked by and/or operably linked to at least 5 contiguous amino acids of an AAV capsid protein. 6. The composition of any one of the preceding claims, wherein the heterologous epitope comprises an amino acid sequence EQKLISEEDL (SEQ ID NO:6) or a portion thereof, and wherein the paratope of the antibody specifically binds to the amino acid sequence EQKLISEEDL or a portion thereof. 7. The composition of claim 1, wherein the nucleotide of interest is: (i) under the control of a promoter selected from the group consisting of a viral promoter, a bacterial promoter, a mammalian promoter, an avian promoter, a fish promoter, an insect promoter, and any combination thereof; optionally wherein the nucleotide of interest is under the control of a non-human promoter; or (ii) under the control of a non-human promoter and flanked by AAV ITR sequences; or (iii) a reporter gene, optionally wherein the reporter gene encodes green fluorescent protein, or is selected from the group consisting of a suicide gene, a nucleotide encoding an antibody or fragment thereof, a nucleotide encoding a CRISPR/Cas system or portion(s) thereof, a nucleotide encoding antisense RNA, a nucleotide encoding siRNA, and a combination thereof. 8. The composition of any one of the preceding claims, wherein the multispecific binding molecule is a bispecific binding molecule. 9. The composition of claim 1, wherein the paratope of the antibody is an Fv domain, optionally wherein the Fv domain is directly fused to a heavy chain constant domain. 10. The composition of claim 1, wherein the retargeting ligand is an antibody or a portion thereof, optionally: wherein the antibody is a bispecific antibody, wherein the antibody paratope and the retargeting ligand each comprise a distinct Fv domain fused to a first and second heavy chain constant domain, optionally wherein the first and second heavy chains bind to protein A with differential binding affinities or wherein the retargeting ligand comprises a tetrameric antibody structure comprising two identical immunoglobulin heavy chains and two identical light chains and wherein the paratope of the antibody that binds to the heterologous epitope is appended to the C-terminus or the N-terminus of one or both heavy chains and/or to the C-terminus or the N-terminus of one or both heavy chains, and optionally wherein the paratope of the antibody is an scFv, wherein, optionally, the scFv comprises a nucleic acid sequence set forth as SEQ ID NO: 37. 11. The composition of any one of the preceding claims, wherein the cell surface protein is a receptor; and/or wherein the retargeting ligand binds to a receptor expressed on the surface of a mammalian or human cell. 12. The composition of any one of the preceding claims, wherein the AAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9 and/or wherein the heterologous epitope is inserted after and/or replaces a position selected from the group consisting of I-453 of AAV2, I-587 of AAV2, I-585 of AAV6, I-590 of AAV8, I-453 of AAV9, I-589 of AAV9 and any corresponding amino acid of a primate-infecting AAV serotype. 13. The composition of any one of the preceding claims, wherein the AAV capsid is a mosaic capsid comprising a reference AAV capsid protein of the same serotype that does not comprise the heterologous epitope in a certain proportion. 14. The composition of any one of the preceding claims, wherein the recombinant AAV capsid protein is derived from a chimeric AAV capsid gene modified to express the heterologous epitope, wherein the chimeric AAV capsid gene comprises a plurality of nucleic acid sequences, wherein each of the plurality of nucleic acid sequences encodes a portion of a capsid protein of a different AAV serotype, and wherein the plurality of nucleic acid sequences together encode a chimeric AAV capsid protein. 15. The composition of any of the preceding claims for use in a method of treatment or diagnosis in vivo, the method comprising administering the nucleotide of interest to a target cell expressing a cell surface protein comprising contacting the target cell with the composition, wherein the multispecific binding molecule comprises a retargeting ligand that binds to the cell surface protein. 16. The composition for use according to claim 15, wherein: (a) the administration is to a human objective; or the target cell is a human cell; or (c) the target cell is a human neuronal cell; or (d) the target cell is a human muscle cell; or (e) the target cell is selected from the group consisting of a liver cell, a brain cell, a T lymphocyte, a kidney cell, an intestinal cell, a pancreas cell, a cancer cell and a cell infected with a heterologous pathogen. 17. The composition for use according to claim 15 or claim 16 wherein: (a) the target cell is a human liver cell optionally wherein the retargeting ligand binds to the human asialoglycoprotein receptor 1 (hASGR1) expressed by the human liver cell; or (b) the retargeting ligand binds to the GABA receptor expressed by the human neuronal cell; or (c) the retargeting ligand binds to the transferrin receptor expressed by the human neuronal cell; (d) the target cell is a human T cell, optionally wherein the retargeting ligand binds to CD3 expressed by the human T cell; or (e) the target cell is a human T cell, optionally wherein the retargeting ligand binds to CD3 expressed by the human T cell; or (f) the target cell is a human hematopoietic cell, optionally wherein the retargeting ligand binds to CD34; or (g) the target cell is a human cancer cell, optionally wherein the retargeting ligand binds to a tumor-associated antigen; or (h) the target cell is a cancer cell, optionally wherein the retargeting ligand binds to a tumor-associated antigen that is E6, E7, or Her2; or (i) the cell surface protein is the human glucagon receptor (hGCGR); or (j) the target cell is an intestinal cell or is a pancreatic cell; optionally wherein the receptor is ENTPD3. 18. The composition for use in the method of claim 15 or 16, wherein: (i) the targeting ligand binds to CD20; either (ii) the retargeting ligand binds to the human glucagon receptor; or (iii) the retargeting ligand binds specifically to CD63 or human ectonucleoside triphosphate diphosphohydrolase 3 (hENTPD3). 19. The composition of claim 5, wherein the affinity tag comprises c-myc (EQKLISEEDL; SEQ ID NO: 6). 20. The composition of claim 6, wherein the antibody paratope specifically binds to c-myc, which antibody paratope comprises a HCVR, LCVR, HCDR1, HCDR2, Hc DR3, LCDR1, LCDR2 and/or LCDR3 sequence of the scFv encoded by the nucleic acid sequence set forth as SEQ ID NO:28. 21. A method of administering a nucleotide of interest in vitro or ex vivo to a target cell expressing a cell surface protein comprising contacting the target cell with the composition of any one of claims 1 to 14, wherein the multispecific binding molecule comprises a retargeting ligand that binds to the cell surface protein.